Method for enhancing proliferation or differentiation of a cell using OB protein

ABSTRACT

Uses for WSX ligands in hematopoiesis are disclosed. In particular, in vitro and in vivo methods for stimulating hematopoiesis (e.g., myelopoiesis, erythropoiesis and especially, lymphopoiesis) using a WSX ligand (e.g., anti-WSX receptor agonist antibodies or OB protein), and optionally another cytokine, are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 08/667,197filed Jun. 20, 1996, which claims priority to U.S. Provisional PatentApplication No. 60/064,855, filed Jan. 8, 1996, hereby incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains generally to the WSX receptor. Inparticular, the invention relates to WSX ligands and uses therefor.

2. Description of the Related Art

Hematopoiesis

The process of blood cell formation whereby red and white blood cellsare replaced through the division of cells located in the bone marrow iscalled hematopoiesis. For a review of hematopoiesis see Dexter andSpooncer (Ann. Rev. Cell Biol. 3:423-441 (1987)).

There are many different types of blood cells which belong to distinctcell lineages. Along each lineage, there are cells at different stagesof maturation. Mature blood cells are specialized for differentfunctions. For example, erythrocytes are involved in O₂ and CO₂transport; T and B lymphocytes are involved in cell and antibodymediated immune responses, respectively; platelets are required forblood clotting; and the granulocytes and macrophages act as generalscavengers and accessory cells. Granulocytes can be further divided intobasophils, eosinophils, neutrophils and mast cells.

Each of the various blood cell types arises from pluripotent ortotipotent stem cells which are able to undergo self-renewal or giverise to progenitor cells or Colony Forming Units (CFU) that yield a morelimited array of cell types. As stem cells progressively lose theirability to self-renew, they become increasingly lineage restricted. Ithas been shown that stem cells can develop into multipotent cells(called “CFC-Mix” by Dexter and Spooncer, supra). Some of the CFC-Mixcells can undergo renewal whereas others lead to lineage-restrictedprogenitors which eventually develop into mature myeloid cells (e.g.,neutrophils, megakaryocytes, macrophages and basophils). Similarly,pluripotent stem cells are able to give rise to PreB and PreT lymphoidcell lineages which differentiate into mature B and T lymphocytes,respectively. Progenitors are defined by their progeny, e.g.,granulocyte/macrophage colony-forming progenitor cells (GM-CFU)differentiate into neutrophils or macrophages; primitive erythroidburst-forming units (BFU-E) differentiate into erythroid colony-formingunits (CFU-E) which give rise to mature erythrocytes. Similarly, theMeg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate intomegakaryocytes, eosinophils and basophils, respectively.

Hematopoietic growth factors (reviewed in D'Andrea, NEJM 330(12):839-846(1994)) have been shown to enhance growth and/or differentiation ofblood cells via activation of receptors present on the surface of bloodprogenitor cells of the bone marrow. While some of these growth factorsstimulate proliferation of restricted lineages of blood cells, othersenhance proliferation of multiple lineages of blood cells. For example,erythropoietin (EPO) supports the proliferation of erythroid cells,whereas interleukin-3 (IL-3) induces proliferation of erythroid andmyeloid lineages and is therefore considered a multi-lineage factor.

In recent years, several hematopoietic growth factor receptors have beenisolated. Due to their low abundance and their existence in bothhigh-affinity and low-affinity forms, biochemical characterization ofthese receptors has been hampered.

Cytokine receptors frequently assemble into multi-subunit complexes.Sometimes, the α subunit of this complex is involved in binding thecognate growth factor and the β-subunit may contain an ability totransduce a signal to the cell. These receptors have been assigned tothree subfamilies depending on the complexes formed. Subfamily 1includes the receptors for erythropoietin (EPO), granulocytecolony-stimulating factor (G-CSF), interleukin-4 (IL-4), interleukin-7(IL-7), growth hormone (GH) and prolactin (PRL). Ligand binding toreceptors belonging to this subfamily is thought to result inhomodimerization of the receptor. Subfamily 2 includes receptors forIL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin-5 (IL-5), interleukin-6 (IL-6), leukemia inhibitory factor(LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF).Subfamily 2 receptors are heterodimers having an α-subunit for ligandbinding and β-subunit (either the shared β-subunit of the IL-3, GM-CSFand IL-5 receptors or the gp130 subunit of the IL-6, LIF, OSM and CNTFreceptors) for signal transduction. Subfamily 3 contains only theinterleukin-2 (IL-2) receptor. The β and γ subunits of the IL-2 receptorcomplex are cytokine-receptor polypeptides which associate with theα-subunit of the unrelated Tac antigen.

Obesity

Obesity is the most common nutritional disorder which, according torecent epidemiologic studies, affects about one third of all Americans20 years of age or older. Kuczmarski et al., J. Am. Med. Assoc.272:205-11 (1994). Obesity is responsible for a variety of serioushealth problems, including cardiovascular disorders, type II diabetes,insulin-resistance, hypertension, hypertriglyceridemia,dyslipoproteinemia, and some forms of cancer. Pi-Sunyer, F., Anns. Int.Med. 119: 655-60 (1993); Colfitz, G., Am. J. Clin. Nutr. 55:503S-507S(1992). A single-gene mutation (the obesity or “ob” mutation) has beenshown to result in obesity and type II diabetes in mice. Friedman,Genomics 11:1054-1062 (1991).

Zhang et al., Nature 372:425-431 (1994) have recently reported thecloning and sequencing of the mouse ob gene and its human homologue, andsuggested that the ob gene product, leptin or OB protein, may functionas part of a signalling pathway from adipose tissue that acts toregulate the size of the body fat depot. Parabiosis experimentsperformed more than 20 years ago predicted that the genetically obesemouse containing two mutant copies of the ob gene (ob/ob mouse) does notproduce a satiety factor which regulates its food intake, while thediabetic (db/db) mouse produces but does not respond to a satietyfactor. Coleman and Hummal, Am. J. Physiol. 217:1298-1304 (1969);Coleman, Diabetol 9:294-98 (1973). Recent reports by three independentresearch teams have demonstrated that daily injections of recombinant OBprotein inhibit food intake and reduce body weight and fat in grosslyobese ob/ob mice but not in db/db mice (Pelleymounter et al., Science269:540-43 (1995); Halaas et al., Science 269:543-46 (1995); Campfieldet al., Science 269: 546-49 (1995)), suggesting that the OB protein issuch a satiety factor as proposed in early cross-circulation studies.

Researchers suggest that at least one OB receptor is localized in thebrain. The identification and expression cloning of a leptin receptor(OB-R) was reported by Tartaglia et al. Cell 83:1263-71 (1995). Variousisoforms of a OB receptor are described by Cioffi et al. Nature 2:585-89(1996). See, also, WO 96/08510.

The mouse db gene has recently been cloned (Lee et al. Nature 379:632(1996) and Chen et al. Cell 84:491-495 (1996)). Previous data hadsuggested that the db gene encoded the receptor for the obese (ob) geneproduct, leptin (Coleman et al., Diebetologia 9:294-8 (1973) and Colemanet al., Diebetologia 14:141-8 (1978)). It has been very recentlyconfirmed that the db/db mouse results from a truncated splice variantof the OB receptor which likely renders the receptor defective in signaltransduction (Lee et al., Nature 379:632 (1996) and Chen et al., Cell84: 491-495 (1996)).

SUMMARY OF THE INVENTION

In one aspect, the present invention pertains to the discovery hereinthat WSX ligands, such as obesity (OB) protein, play a role inhematopoiesis via signalling through the WSX receptor. The role of theWSX receptor-ligand signalling pathway appears to be at the level of theearly hematopoietic precursor as is evident by the ability of OB proteinto simulate myelopoiesis, erythropoiesis (e.g. splenic erythropoiesis)and most dramatically, lymphopoiesis. Accordingly, WSX ligands can beused to stimulate proliferation and/or differentiation and/or survivalof hematopoietic progenitor cells either in vitro or in vivo (e.g. fortreating hematopoietic diseases or disorders).

Thus, the invention provides a method for stimulating proliferationand/or differentiation of a cell which expresses the WSX receptor(especially the WSX receptor variant 13.2, which is demonstrated hereinto have the capacity to transmit a proliferative signal) at its cellsurface comprising the step of contacting the WSX receptor with anamount of WSX ligand which is effective for stimulating proliferationand/or OB protein differentiation of the cell. In preferred embodiments,the cell which is exposed to the WSX ligand is a hematopoeiticprecursor, e.g. a CD34+ cell. The WSX ligand may be OB protein or anagonist antibody which binds to the WSX receptor. For in vivo use, theWSX ligand of choice may be a long half-life derivative of an OBprotein, such as OB-immunoglobulin chimera and/or OB protein modifiedwith a nonproteinaceous polymer, such as polyethylene glycol (PEG). Themethod contemplated herein may lead to an increase in the proliferationand/or differentiation of lymphoid, myeloid and/or erythroid blood celllineages and encompasses both in vitro and in vivo methods. For in vitrouses, the cell possessing the WSX receptor may be present in cellculture. As to in vivo methods, the cell may be present in a mammal,especially a human (e.g. one who is suffering from decreased bloodlevels and who could benefit from an increase in various blood cells).Potential patients include those who have undergone chemo- or radiationtherapy, or bone marrow transplantation therapy. Thus, the inventionprovides a method for repopulating blood cells (e.g. erythroid, myeloidand/or lymphoid blood cells) in a mammal comprising administering to themammal a therapeutically effective amount of a WSX ligand.

Mammals which may benefit from an enhancement of lymphopoiesis includethose predisposed to, or suffering from, any one or more of thefollowing exemplary conditions: lymphocytopenia; lymphorrhea;lymphostasis; immunodeficiency (e.g. HIV and AIDS); infections(including, for example, opportunistic infections and tuberculosis(TB)); lupus; and other disorders characterized by lymphocytedeficiency. An effective amount of the WSX ligand can be used in amethod of immunopotentiation or to improve immune function in a mammal.

On the other hand, WSX receptor or WSX ligand antagonists (such as WSXreceptor ECD or immunoadhesin, and WSX receptor or OB proteinneutralizing antibodies) may be used in the treatment of those disorderswherein unacceptable lymphocyte levels are present in the mammal,particularly where this is caused by excessive activation of the WSXreceptor. Examples of conditions in which administration of such anantagonist may be beneficial include: neoplastic disorders (such asHodkin's disease; lymphosarcoma; lymphoblastoma; lymphocytic leukemia;and lymphoma) and lymphocytosis.

Diseases or disorders in which an increase in erythropoiesis may bebeneficial include, but are not limited to: erythrocytopenia;erthrodegenerative disorders; erythroblastopenia; leukoerythroblastosis;erythroclasis; thalassemia; and anemia (e.g. hemolytic anemia, such asacquired, autoimmune, or microangiopathic hemolytic anemia; aplasticanemia; congenital anemia, e.g., congenital dyserythropoietic anemia,congenital hemolytic anemia or congenital hypoplastic anemia;dyshemopoietic anemia; Faconi's anemia; genetic anemia; hemorrhagicanemia; hyperchromic or hypochromic anemia; nutritional, hypoferric, oriron deficiency anemia; hypoplastic anemia; infectious anemia; leadanemia; local anemia; macrocytic or microcytic anemia; malignant orpernicious anemia; megaloblastic anemia; molecular anemia; normocyticanemia; physiologic anemia; traumatic or posthemorrhagic anemia;refractory anemia; radiation anemia; sickle cell anemia; splenic anemia;and toxic anemia).

Conversely, WSX receptor or WSX ligand antagonists may be used to treatthose conditions in which excessive erythrocyte levels are present in amammal, e.g. in neoplastic disorders such as erythroleukemia;erythroblastosis; and erythrocythemia or polycythemia).

An increase in myelopoiesis may be beneficial in any of theabove-mentioned diseases or disorders as well as the following exemplaryconditions: myelofibrosis; thrombocytopenia; hypoplasia; disseminatedintravascular coagulation (DIC); immune (autoimmune) thrombocytopenicpurpura (ITP); HIV induced ITP; myelodysplasia; thrombocytotic diseasesand thrombocytosis.

Antagonists of the WSX receptor-WSX ligand interaction may also be usedto treat myeloid cell-related conditions such as malignancies (e.g.myelosarcoma, myeloblastoma, myeloma, myeloleukemia andmyelocytomatosis); myeloblastosis; myelocytosis; and myelosis.

The method may further involve the step of exposing hematopoeitic cells(whether they be in cell culture or in a mammal) to one or more othercytokines (e.g. lineage-specific cytokines) and this may lead to asynergistic enhancement of the proliferation and/or differentiation ofthe cells. Exemplary cytokines include thrombopoietin (TPO);erythropoietin (EPO); macrophage-colony stimulating factor (M-CSF);granulocyte-macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF);interleukin-1 (IL-1); IL-1α; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8;IL-9; IL-11; IL-10; IL-12; leukemia inhibitory factor (LIF) or kitligand (KL). In this embodiment, exposure to the cytokine may proceed,occur simultaneously with, or follow, exposure to the WSX ligand.Preferably, the WSX ligand and one or more further cytokines areadministered simultaneously to the patient (where the method is an invivo one) and, optionally, are combined to form a pharmaceuticalcomposition.

For use in the above methods, the invention also provides an article ofmanufacture, comprising: a container; a label on the container; and acomposition comprising an active agent within the container; wherein thecomposition is effective for enhancing proliferation and/ordifferentiation of cells comprising the WSX receptor in a mammal, thelabel on the container indicates that the composition can be used forenhancing proliferation and/or differentiation of those cells and theactive agent in the composition is a WSX ligand. Optionally, the articleof manufacture includes one or more futher containers which hold furthercytokine(s) in a packaged combination with the container holding the WSXligand.

In another embodiment, an effective amount of the WSX ligand may be usedto improve engraftment in bone marrow transplantation or to stimulatemobilization of hematopoietic stem cells in a mammal prior to harvestinghematopoietic progenitors from the peripheral blood thereof.

According to a further aspect, the invention is concerned with the WSXcytokine receptor and a soluble form of the receptor which is the WSXreceptor extracellular domain (ECD). The WSX receptor polypeptides areoptionally conjugated with, or fused to, molecules which increase theserum half-lives thereof and can be formulated as pharmaceuticalcompositions comprising the polypeptide and a physiologically acceptablecarrier.

In certain embodiments, the WSX receptor ECD may be used as anantagonist insofar as it may bind to WSX ligand and thereby reduceactivation of endogenous WSX receptor. This may be useful in conditionscharacterized by excess levels of WSX ligand and/or excess WSX receptoractivation in a mammal. WSX receptor ECD may, for example, be used totreat metabolic disorders (e.g., anorexia or steroid-inducedtruncalobesity), stem cell tumors and other tumors which express WSXreceptor.

Pharmaceutical compositions of the WSX receptor ECD may further includea WSX ligand. Such dual compositions may be beneficial where it istherapeutically useful to prolong the half-life of WSX ligand and/oractivate endogenous WSX receptor directly as a heterotrimeric complex.

The invention also relates to chimeric WSX receptor molecules, such asWSX receptor immunoadhesins (having long half-lives in the serum of apatient treated therewith) and epitope tagged WSX receptor.Immunoadhesins may be employed as WSX receptor antagonists in conditionsor disorders in which neutralization of WSX receptor biological activitymay be beneficial. Bispecific immunoadhesins (combining a WSX receptorECD with a domain of another cytokine receptor) may form high affinitybinding complexes for WSX ligand.

The invention further provides methods for identifying a molecule whichbinds to and/or activates the WSX receptor. This is useful fordiscovering molecules (such as peptides, antibodies, and smallmolecules) which are agonists or antagonists of the WSX receptor. Suchmethods generally involve exposing an immobilized WSX receptor to amolecule suspected of binding thereto and determining binding of themolecule to the immobilized WSX receptor and/or evaluating whether ornot the molecule activates (or blocks activation of) the WSX receptor.In order to identify such WSX ligands, the WSX receptor may be expressedon the surface of a cell and used to screen libraries of syntheticcompounds and naturally occurring compounds (e.g., endogenous sources ofsuch naturally occurring compounds, such as serum). The WSX receptor canalso be used as a diagnostic tool for measuring serum levels ofendogenous WSX ligand.

In a further embodiment, a method for purifying a molecule which bindsto the WSX receptor is provided. This can be used in the commercialproduction and purification of therapeutically active molecules whichbind to this receptor. In the method, the molecule of interest(generally a composition comprising one or more contaminants) isadsorbed to immobilized WSX receptor (e.g., WSX receptor immunoadhesinimmobilized on a protein A column). The contaminants, by virtue of theirinability to bind to the WSX receptor, will generally flow through thecolumn. Accordingly, it is then possible to recover the molecule ofinterest from the column by changing the elution conditions, such thatthe molecule no longer binds to the immobilized receptor.

In further embodiments, the invention provides antibodies thatspecifically bind to the WSX receptor. Preferred antibodies aremonoclonal antibodies which are non-immunogenic in a human and bind toan epitope in the extracellular domain of the receptor. Preferredantibodies bind the WSX receptor with an affinity of at least about 10⁶L/mole, more preferably 10⁷ L/mole.

Antibodies which bind to the WSX receptor may optionally be fused to aheterologous polypeptide and the antibody or fusion thereof may be usedto isolate and purify WSX receptor from a source of the receptor.

In a further aspect, the invention provides a method for detecting theWSX receptor in vitro or in vivo comprising contacting the antibody witha sample suspected of containing the receptor and detecting if bindinghas occurred. Based on the observation herein that CD34+ cells possessWSX receptor, use of WSX antibodies for identification and/or enrichmentof stem cell populations (in a similar manner to that in which CD34antibodies are presently used) is envisaged.

For certain applications, it is desirable to have an agonist antibodywhich can be screened for as described herein. Such agonist antibodiesare useful for activating the WSX receptor for in vitro uses wherebyenhancement of proliferation and/or differentiation of a cell comprisingthe receptor is desired. Furthermore, these antibodies may be used totreat conditions in which an effective amount of WSX receptor activationleads to a therapeutic benefit in the mammal treated therewith. Forexample, the agonist antibody can be used to enhance survival,proliferation and/or differentiation of a cell comprising the WSXreceptor. In particular, agonist antibodies and other WSX ligands may beused to stimulate proliferation of stem cells/progenitor cells either invitro or in vivo. Other potential therapeutic applications include theuse of agonist antibodies to treat metabolic disorders (such as obesityand diabetes) and to promote kidney, liver or lung growth and/or repair(e.g., in renal failure).

For therapeutic applications it is desirable to prepare a compositioncomprising the agonist antibody and a physiologically acceptablecarrier. Optionally, such a composition may further comprise one or morecytokines.

In other embodiments, the antibody is a neutralizing antibody. Suchmolecules can be used to treat conditions characterized by unwanted orexcessive activation of the WSX receptor.

In addition to the above, the invention provides isolated nucleic acidmolecules, expression vectors and host cells encoding the WSX receptorwhich can be used in the recombinant production of WSX receptor asdescribed herein. The isolated nucleic acid molecules and vectors arealso useful for gene therapy applications to treat patients with WSXreceptor defects and/or to increase responsiveness of cells to WSXligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-J together depict the double stranded nucleotide (SEQ ID NO:1)and deduced amino acid sequence (SEQ ID NO:2) encoding full length humanWSX receptor variant 13.2. Nucleotides are numbered at the beginning ofthe sense strand. Amino acid residues are numbered at the beginning ofthe amino acid sequence. Restriction enzyme sites are depicted above thenucleotide sequence.

FIGS. 2A-D together depict an amino acid sequence alignment of fulllength human WSX receptor variants 6.4 (SEQ ID NO:3), 12.1 (SEQ ID NO:4)and 13.2, respectively. Homologous residues are boxed. WSX receptorvariants 6.4, 12.1 and 13.2 are native sequence human WSX receptorvariants which, without being bound to any one theory, appear to begenerated by alternate splicing of WSX receptor mRNA. The putativesignal peptide, transmembrane, Box 1, Box 2, and Box 3 domains areindicated. The extracellular and cytoplasmic domains are amino- andcarboxy-terminal, respectively, to the transmembrane domain. The Box 1-3domains shown correspond to the box 1-3 motifs described in Baumann etal., Mol. Cell. Biol. 14(1):138-146 (1994).

FIGS. 3A-L together depict an alignment of the nucleotide sequencesencoding human WSX receptor variants 6.4 (SEQ ID NO:5), 12.1 (SEQ IDNO:6) and 13.2, respectively.

FIGS. 4A-D depict an alignment of the full length human WSX receptorvariant 13.2 amino acid sequence (top) with that of partial murine WSXreceptor extracellular domain sequence (bottom) (SEQ ID NO:7) obtainedas described in Example 7. The putative murine signal peptide is markedwith an arrow.

FIGS. 5A-M represent an alignment of the nucleotide sequences encodinghuman WSX receptor variant 13.2 (bottom) and partial murine WSX receptorextracellular domain (top) (SEQ ID NO:8), respectively.

FIG. 6 is a bar graph depicting results of the thymidine incorporationassay described in Example 5. ³H-thymidine incorporation (counts perminute, CPM) in parental Baf3 cells or Baf3 cells electroporated withGH/WSX variant 13.2 chimera in the presence of varying concentrations ofhuman growth hormone (GH) is shown.

FIG. 7 shows the human and murine oligonucleotides (SEQ ID NOS:9-38,respectively) used for the antisense experiment described in Example 8.

FIGS. 8 and 9 show thymidine incorporation assays in Baf-3 cells. Forthese assays, cells were deprived of IL-3 for 16-18 hours (in RPMI 1640supplemented with 10% fetal calf serum (FCS)). Cells were washed inserum free RPMI 1640 and plated at 50,000 cells per well in 0.2 mls ofserum free RPMI 1640 supplemented with the indicated concentration ofhuman GH or human OB protein. Cells were stimulated for 24 hours andthymidine incorporation was determined as described (Zeigler et al.Blood 84:2422-2430. (1994)). Assays were performed in triplicate and theresults were confirmed in three independent experiments.

In FIG. 8, GH receptor-WSX receptor variant 12.1 or 13.2 chimericproteins were expressed in Baf-3 cells as described in Example 5. Thesetransfected cells and the parental Baf-3 line were stimulated with hGHand the incorporation of titrated thymidine determined.

In FIG. 9, Baf-3 cells were stably transfected with WSX receptor variant13.2. Thymidine incorporation was then determined in these cell linesfollowing stimulation with human OB protein.

In FIGS. 10A-C, murine fetal liver AA4⁺Sca⁺Kit⁺ (flASK) stem cells werecultured in suspension culture or methylcellulose. In FIG. 10A, flASKcells were cultured in suspension culture containing serum with kitligand (KL) or kit ligand and OB protein. Cell counts and cytospinanalyses were performed 7 days later. In FIG. 10B, flASK cells wereseeded into methylcellulose under either myeloid or lymphoid conditionsas described in Example 10. Colony counts were performed 14 days later.For colonies produced under lymphoid conditions, FACS analysisdemonstrated the vast majority of cells to be B220 positive. In FIG.10C, flASK cells were seeded into methylcellulose containing kit ligand.To this base media, erythropoietin (EPO) or erythropoietin and OBprotein were then added. The resultant colonies were counted 14 dayslater. FACS analysis demonstrated approximately 95% of these colonies tobe TER 119 positive. All assays were performed in triplicate andconfirmed in at least three independent experiments.

FIG. 11 illustrates methylcellulose assays to determine the colonyforming potential of db/db, ob/ob and the corresponding wild-typemarrow. 100,000 bone marrow cells were seeded into methylcellulose andthe resultant colonies counted after 14 days. Assays were performedusing both myeloid and lymphoid conditions. Assays were performed intriplicate and the experiments were repeated a minimum of 3 times.

FIGS. 12A-B show bone marrow cellular profiles in wild-type misty grayhomozygotes, misty gray/db heterozygotes, and homozygote db/db mice.Overall cellularity in the db/db marrow was unchanged compared tocontrols. FIG. 12A shows cellular profiles determined using anti-B220,anti-CD43, and anti-TER119 antibodies. FIG. 12B shows cellular profilesof the spleens from the above groups.

FIGS. 13A-C are an analysis of peripheral blood in db/db homozygotes,db/db misty gray heterozygotes and misty gray homozygotes. 40microliters of peripheral blood was taken via orbital bleed and analyzedon a Serrono Baker system 9018. All areas described by the boxesrepresent the mean±one standard deviation of the two parameters.

FIG. 14 is a comparison of peripheral lymphocyte counts and bloodglucose level. Five groups of animals, misty-gray, misty-gray/db, db/db,interferon α-transgenic, and glucokinase transgenic heterozygote mice(gLKa) were sampled via retro-orbital bleed. Blood glucose levels inthese mice were determined. All areas described by the boxes representthe mean±standard deviation of the two parameters.

In FIGS. 15A-C, misty gray homozygotes, db/misty gray heterozygotes, andhomozygous db/db mice were subjected to sub-lethal irradiation and therecovery kinetics of the peripheral blood was determined viaretro-orbital bleeds.

FIGS. 16A-16V together show the nucleotide sequence (SEQ ID NO:46) andthe amino acid sequence (SEQ ID NO: 47) of the human OB-immunoglobulinchimera in the plasmid described in of Example 11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “WSX receptor” or “WSX receptor polypeptide” when used hereinencompass native sequence WSX receptor; WSX receptor variants; WSXextracellular domain; and chimeric WSX receptor (each of which isdefined herein). Optionally, the WSX receptor is not associated withnative glycosylation. “Native glycosylation” refers to the carbohydratemoieties which are covalently attached to WSX receptor when it isproduced in the mammalian cell from which it is derived in nature.Accordingly, human WSX receptor produced in a non-human cell is anexample of a WSX receptor which is “not associated with nativeglycosylation”. Sometimes, the WSX receptor is unglycosylated (e.g., asa result of being produced recombinantly in a prokaryote).

“WSX ligand” is a molecule which binds to and activates native sequenceWSX receptor (especially WSX receptor variant 13.2). The ability of amolecule to bind to WSX receptor can be determined by the ability of aputative WSX ligand to bind to WSX receptor immunoadhesin (see Example2) coated on an assay plate, for example. The thymidine incorporationassay provides a means for screening for WSX ligands which activate theWSX receptor. Exemplary WSX ligands include anti-WSX receptor agonistantibodies and OB protein (e.g., described in Zhang et al. Nature372:425-431 (1994)).

The terms “OB protein” and “OB” are used interchangeably herein andrefer to native sequence OB proteins (also known as “leptins”) and theirfunctional derivatives.

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., WSX receptor or OB protein) derivedfrom nature. Such native sequence polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. Thus, anative sequence polypeptide can have the amino acid sequence ofnaturally occurring human polypeptide, murine polypeptide, orpolypeptide from any other mammalian species.

The term “native sequence WSX receptor” specifically encompassesnaturally-occurring truncated forms of the WSX receptor,naturally-occurring variant forms (e.g., alternatively spliced formssuch as human WSX receptor variants 6.4, 12.1 and 13.2 described herein)and naturally-occurring allelic variants of the WSX receptor. Thepreferred native sequence WSX receptor is a mature native sequence humanWSX receptor, such as human WSX receptor variant 6.4, human WSX receptorvariant 12.1 or human WSX receptor variant 13.2 (each shown in FIGS.2A-D). Most preferred is mature human WSX receptor variant 13.2.

The term “native sequence OB protein” includes those OB proteins fromany animal species (e.g. human, murine, rabbit, cat, cow, sheep,chicken, porcine, equine, etc.) as occurring in nature. The definitionspecifically includes variants with or without a glutamine at amino acidposition 49, using the amino acid numbering of Zhang et al., supra. Theterm “native sequence OB protein” includes the native proteins with orwithout the initiating N-terminal methionine (Met), and with or withoutthe native signal sequence, either in monomeric or in dimeric form. Thenative sequence human and murine OB proteins known in the art are 167amino acids long, contain two conserved cysteines, and have the featuresof a secreted protein. The protein is largely hydrophilic, and thepredicted signal sequence cleavage site is at position 21, using theamino acid numbering of Zhang et al., supra. The overall sequencehomology of the human and murine sequences is about 84%. The twoproteins show a more extensive identity in the N-terminal region of themature protein, with only four conservative and three non-conservativesubstitutions among the residues between the signal sequence cleavagesite and the conserved Cys at position 117. The molecular weight of OBprotein is about 16 kD in a monomeric form.

The “WSX receptor extracellular domain” (ECD) is a form of the WSXreceptor which is essentially free of the transmembrane and cytoplasmicdomains of WSX receptor, i.e., has less than 1% of such domains,preferably 0.5 to 0% of such domains, and more preferably 0.1 to 0% ofsuch domains. Ordinarily, the WSX receptor ECD will have an amino acidsequence having at least about 95% amino acid sequence identity with theamino acid sequence of the ECD of WSX receptor indicated in FIGS. 2A-Dfor human WSX receptor variants 6.4, 12.1 and 13.2, preferably at leastabout 98%, more preferably at least about 99% amino acid sequenceidentity, and thus includes WSX receptor variants as defined below.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide (e.g., WSX receptor having the deduced amino acidsequence shown in FIGS. 1A-J for human WSX receptor variant 13.2). Suchvariants include polypeptides wherein one or more amino acid residuesare added at the N- or C-terminus of, or within, the native sequence;from about one to thirty amino acid residues are deleted, and optionallysubstituted by one or more amino acid residues; and derivatives of theabove polypeptides, wherein an amino acid residue has been covalentlymodified so that the resulting product has a non-naturally occurringamino acid. Ordinarily, a biologically active WSX receptor variant willhave an amino acid sequence having at least about 90% amino acidsequence identity with human WSX receptor variant 13.2 shown in FIGS.1A-J, preferably at least about 95%, more preferably at least about 99%.Ordinarily, a biologically active OB protein variant will have an aminoacid sequence having at least about 90% amino acid sequence identitywith a native sequence OB protein, preferably at least about 95%, morepreferably at least about 99%.

A “chimeric” OB protein or WSX receptor is a polypeptide comprising OBprotein or full-length WSX receptor or one or more domains thereof(e.g., the extracellular domain of the WSX receptor) fused or bonded toheterologous polypeptide. The chimeric WSX receptor will generally shareat least one biological property in common with human WSX receptorvariant 13.2. The chimeric OB protein will generally share at least onebiological property in common with a native sequence OB protein.Examples of chimeric polypeptides include immunoadhesins and epitopetagged polyeptides.

The term “WSX immunoadhesin” is used interchangeably with the expression“WSX receptor-immunoglobulin chimera” and refers to a chimeric moleculethat combines a portion of the WSX receptor (generally the extracellulardomain thereof) with an immunoglobulin sequence. Likewise, an “OBprotein immunoadhesin” or “OB-immunoglobulin chimera” refers to achimeric molecule which combines OB protein (or a portion thereof) withan immunoglobulin sequence. The immunoglobulin sequence preferably, butnot necessarily, is an immunoglobulin constant domain. Theimmunoglobulin moiety in the chimeras of the present invention may beobtained from IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM,but preferably IgG1 or IgG3.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising WSX receptor or OB protein fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody thereagainst can be made, yet is shortenough such that it does not interfere with biological activity of theWSX receptor or OB protein. The tag polypeptide preferably also isfairly unique so that the antibody thereagainst does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least six amino acid residues and usually between about 8-50amino acid residues (preferably between about 9-30 residues).

“Isolated” WSX receptor (or OB protein) means WSX receptor (or OBprotein) that has been purified from a WSX receptor (or OB protein)source or has been prepared by recombinant or synthetic methods and issufficiently free of other peptides or proteins (1) to obtain at least15 and preferably 20 amino acid residues of the N-terminal or of aninternal amino acid sequence by using a spinning cup sequenator or thebest commercially available amino acid sequenator marketed or asmodified by published methods as of the filing date of this application,or (2) to homogeneity by SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins.

“Essentially pure” protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight. “Essentially homogeneous”protein means a composition comprising at least about 99% by weight ofprotein, based on total weight of the composition.

“Biological property” when used in conjunction with either “WSXreceptor” or “isolated WSX receptor” means having an effector orantigenic function or activity that is directly or indirectly caused orperformed by native sequence WSX receptor (whether in its native ordenatured conformation). Effector functions include ligand binding; andenhancement of survival, differentiation and/or proliferation of cells(especially proliferation of cells). However, effector functions do notinclude possession of an epitope or antigenic site that is capable ofcross-reacting with antibodies raised against native sequence WSXreceptor.

“Biological property” when used in conjunction with either “OB protein”or “isolated OB protein” means having an effector function that isdirectly or indirectly caused or performed by native sequence OBprotein. Effector functions of native sequence OB protein include WSXreceptor binding and activation; and enhancement of differentiationand/or proliferation of cells expressing this receptor (as determined inthe thymidine incorporation assay, for example). A “biologically active”OB protein is one which possesses a biological property of nativesequence OB protein.

A “functional derivative” of a native sequence OB protein is a compoundhaving a qualitative biological property in common with a nativesequence OB protein. “Functional derivatives” include, but are notlimited to, fragments of native sequence OB proteins and derivatives ofnative sequence OB proteins and their fragments, provided that they havea biological activity in common with a corresponding native sequence OBprotein. The term “derivative” encompasses both amino acid sequencevariants of OB protein and covalent modifications thereof.

The phrase “long half-life” as used in connection with OB derivatives,concerns OB derivatives having a longer plasma half-life and/or slowerclearance than a corresponding native sequence OB protein. The longhalf-life derivatives preferably will have a half-life at least about1.5-times longer than a native OB protein; more preferably at leastabout 2-times longer than a native OB protein, more preferably at leastabout 3-time longer than a native OB protein. The native OB proteinpreferably is that of the individual to be treated.

An “antigenic function” means possession of an epitope or antigenic sitethat is capable of cross-reacting with antibodies raised against nativesequence WSX receptor. The principal antigenic function of a WSXreceptor is that it binds with an affinity of at least about 10⁶ L/moleto an antibody raised against native sequence WSX receptor. Ordinarily,the polypeptide binds with an affinity of at least about 10⁷ L/mole. Theantibodies used to define “antigenic function” are rabbit polyclonalantibodies raised by formulating the WSX receptor in Freund's completeadjuvant, subcutaneously injecting the formulation, and boosting theimmune response by intraperitoneal injection of the formulation untilthe titer of the anti-WSX receptor or antibody plateaus.

“Biologically active” when used in conjunction with either “WSXreceptor” or “isolated WSX receptor” means a WSX receptor polypeptidethat exhibits or shares an effector function of native sequence WSXreceptor and that may (but need not) in addition possess an antigenicfunction. A principal effector function of the WSX receptor is itsability to induce proliferation of CD34+human umbilical cord blood cellsin the colony assay described in Example 8.

“Antigenically active” WSX receptor is defined as a polypeptide thatpossesses an antigenic function of WSX receptor and that may (but neednot) in addition possess an effector function.

“Percent amino acid sequence identity” is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the native sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into thecandidate sequence shall be construed as affecting sequence identity orhomology.

A “thymidine incorporation assay” can be used to screen for moleculeswhich activate the WSX receptor. In order to perform this assay, IL-3dependent Baf3 cells (Palacios et al., Cell, 41:727-734 (1985)) arestably transfected with full length native sequence WSX receptor asdescribed in Example 4. The WSX receptor/Baf3 cells so generated arestarved of IL-3 for, e.g., 24 hours in a humidified incubator at 37° C.in 5% CO₂ and air. Following IL-3 starvation, the cells are plated outin 96 well culture dishes with, or without, a test sample containing apotential agonist (such test samples are optionally diluted) andcultured for 24 hours in a cell culture incubator. 20 μl of serum freeRPMI media containing 1 μCi of ³H thymidine is added to each well forthe last 6-8 hours. The cells are then harvested in 96 well filterplates and washed with water. The filters are then counted using aPackard Top Count Microplate Scintillation Counter, for example.Agonists are expected to induce a statistically significant increase (toa P value of 0.05) in ³H uptake, relative to control. Preferred agonistsleads to an increase in ³H uptake which is at least two fold of that ofthe control.

An “isolated” WSX receptor nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the WSX receptor nucleic acid. An isolated WSXreceptor nucleic acid molecule is other than in the form or setting inwhich it is found in nature. Isolated WSX receptor nucleic acidmolecules therefore are distinguished from the WSX receptor nucleic acidmolecule as it exists in natural cells. However, an isolated WSXreceptor nucleic acid molecule includes WSX receptor nucleic acidmolecules contained in cells that ordinarily express WSX receptor where,for example, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies, antibody compositions with polyepitopicspecificity, bispecific antibodies, diabodies, and single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv),so long as they exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 (Cabilly etal.)). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597(1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Thehumanized antibody includes a Primatized™ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

“Non-immunogenic in a human” means that upon contacting the polypeptideof interest in a physiologically acceptable carrier and in atherapeutically effective amount with the appropriate tissue of a human,no state of sensitivity or resistance to the polypeptide of interest isdemonstrable upon the second administration of the polypeptide ofinterest after an appropriate latent period (e.g., 8 to 14 days).

By “agonist antibody” is meant an antibody which is able to activatenative sequence WSX receptor. WSX receptor activation can be determinedusing the thymidine incorporation assay described above.

A “neutralizing antibody” is one which is able to block or significantlyreduce an effector function of native sequence WSX receptor or OBprotein. For example, a neutralizing antibody may inhibit or reduce WSXreceptor activation by a WSX ligand as determined in the thymidineincorporation assay.

An “antagonist” of the WSX receptor and/or OB protein is a moleculewhich prevents, or interferes with, binding and/or activation of the WSXreceptor or OB protein. Such molecules can be screened for their abilityto competitively inhibit WSX receptor activation by OB protein in thethymidine incorporation assay disclosed herein, for example. Examples ofsuch molecules include: WSX receptor ECD; WSX receptor immunoadhesin;neutralizing antibodies against WSX receptor or OB protein; smallmolecule and peptide antagonists; and antisense nucleotides against theWSX receptor or ob gene.

The phrase “enhancing proliferation of a cell” encompasses the step ofincreasing the extent of growth and/or reproduction of the cell relativeto an untreated cell either in vitro or in vivo. An increase in cellproliferation in cell culture can be detected by counting the number ofcells before and after exposure to a molecule of interest. The extent ofproliferation can be quantified via microscopic examination of thedegree of confluency. Cell proliferation can also be quantified usingthe thymidine incorporation assay described herein.

By “enhancing differentiation of a cell” is meant the act of increasingthe extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g., identifying morphologicalchanges in the cell).

A “hematopoietic progenitor cell” or “primitive hematopoietic cell” isone which is able to differentiate to form a more committed or matureblood cell type.

“Lymphoid blood cell lineages” are those hematopoietic precursor cellswhich are able to differentiate to form lymphocytes (B-cells orT-cells). Likewise, “lymphopoeisis” is the formation of lymphocytes.

“Erythroid blood cell lineages” are those hematopoietic precursor cellswhich are able to differentiate to form erythrocytes (red blood cells)and “erythropoeisis” is the formation of erythrocytes.

The phrase “myeloid blood cell lineages”, for the purposes herein,encompasses all hematopoietic precursor cells, other than lymphoid anderythroid blood cell lineages as defined above, and “myelopoiesis”involves the formation of blood cells (other than lymphocytes anderythrocytes).

A “CD34+ cell population” is enriched for hematopoietic stem cells. ACD34+ cell population can be obtained from umbilical cord blood or bonemarrow, for example. Human umbilical cord blood CD34+ cells can beselected for using immunomagnetic beads sold by Miltenyi (California),following the manufacturer's directions.

“Physiologically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, Pluronics or polyethylene glycol (PEG).

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, andIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. Exemplary salvage receptor binding epitope sequencesinclude HQNLSDGK (SEQ ID NO:39); HQNISDGK (SEQ ID NO:40); HQSLGTQ (SEQID NO:41); VISSHLGQ (SEQ ID NO:42); and PKNSSMISNTP (SEQ ID NO:43).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are OB protein;growth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such asTGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin(EPO); osteoinductive factors; interferons such as interferon-α, -β, and-γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; and other polypeptide factors includingleukemia inhibitory factor (LIF) and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

A “lineage-specific cytokine” is one which acts on relatively committedcells in the hematopoietic cascade and gives rise to an expansion inblood cells of a single lineage. Examples of such cytokines include EPO,TPO, and G-CSF.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The term “obesity” is used to designate a condition of being overweightassociated with excessive bodily fat. The desirable weight for a certainindividual depends on a number of factors including sex, height, age,overall built, etc. The same factors will determine when an individualis considered obese. The determination of an optimum body weight for agiven individual is well within the skill of an ordinary physician.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

By “solid phase” is meant a non-aqueous matrix to which a reagent ofinterest (e.g., the WSX receptor or an antibody thereto) can adhere.Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g., an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

Modes for Carrying Out the Invention

The present invention is based on the discovery of the WSX receptor. Theexperiments described herein demonstrate that this molecule is acytokine receptor which appears to play a role in enhancingproliferation and/or differentiation of hematopoietic cells. Inparticular, this receptor has been found to be present in enriched humanstem cell populations, thus indicating that WSX ligands, such as agonistantibodies, may be used to stimulate proliferation of hematopoietic stemcells/progenitor cells. Other uses for this receptor will be apparentfrom the following discussion. A description follows as to how WSXreceptor or OB proteins may be prepared.

A. Preparation of WSX Receptor or OB Protein

Techniques suitable for the production of WSX receptor or OB protein arewell known in the art and include isolating WSX receptor or OB proteinfrom an endogenous source of the polypeptide, peptide synthesis (using apeptide synthesizer) and recombinant techniques (or any combination ofthese techniques). The preferred technique for production of WSXreceptor or OB protein is a recombinant technique to be described below.

Most of the discussion below pertains to recombinant production of WSXreceptor or OB protein by culturing cells transformed with a vectorcontaining WSX receptor or OB protein nucleic acid and recovering thepolypeptide from the cell culture. It is further envisioned that the WSXreceptor or OB protein of this invention may be produced by homologousrecombination, as provided for in WO 91/06667, published 16 May 1991.

Briefly, this method involves transforming primary human cellscontaining a WSX receptor or OB protein-encoding gene with a construct(i.e., vector) comprising an amplifiable gene (such as dihydrofolatereductase (DHFR) or others discussed below) and at least one flankingregion of a length of at least about 150 bp that is homologous with aDNA sequence at the locus of the coding region of the WSX receptor or OBprotein gene to provide amplification of the WSX receptor or OB proteingene. The amplifiable gene must be at a site that does not interferewith expression of the WSX receptor or OB protein gene. Thetransformation is conducted such that the construct becomes homologouslyintegrated into the genome of the primary cells to define an amplifiableregion.

Primary cells comprising the construct are then selected for by means ofthe amplifiable gene or other marker present in the construct. Thepresence of the marker gene establishes the presence and integration ofthe construct into the host genome. No further selection of the primarycells need be made, since selection will be made in the second host. Ifdesired, the occurrence of the homologous recombination event can bedetermined by employing PCR and either sequencing the resultingamplified DNA sequences or determining the appropriate length of the PCRfragment when DNA from correct homologous integrants is present andexpanding only those cells containing such fragments. Also if desired,the selected cells may be amplified at this point by stressing the cellswith the appropriate amplifying agent (such as methotrexate if theamplifiable gene is DHFR), so that multiple copies of the target geneare obtained. Preferably, however, the amplification step is notconducted until after the second transformation described below.

After the selection step, DNA portions of the genome, sufficiently largeto include the entire amplifiable region, are isolated from the selectedprimary cells. Secondary mammalian expression host cells are thentransformed with these genomic DNA portions and cloned, and clones areselected that contain the amplifiable region. The amplifiable region isthen amplified by means of an amplifying agent if not already amplifiedin the primary cells. Finally, the secondary expression host cells nowcomprising multiple copies of the amplifiable region containing WSXreceptor or OB protein are grown so as to express the gene and producethe protein.

1. Isolation of DNA Encoding WSX Receptor or OB Protein

The DNA encoding WSX receptor or OB protein may be obtained from anycDNA library prepared from tissue believed to possess the WSX receptoror OB protein mRNA and to express it at a detectable level. Accordingly,WSX receptor or OB protein DNA can be conveniently obtained from a cDNAlibrary prepared from mammalian fetal liver. The WSX receptor or OBprotein-encoding gene may also be obtained from a genomic library or byoligonucleotide synthesis.

Libraries are screened with probes (such as antibodies to the WSXreceptor or OB protein, or oligonucleotides of about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989). An alternative means to isolatethe gene encoding WSX receptor or OB protein is to use PCR methodologyas described in section 14 of Sambrook et al., supra.

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioushuman tissues, preferably human fetal liver. The oligonucleotidesequences selected as probes should be of sufficient length andsufficiently unambiguous that false positives are minimized.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ³²P-labeled ATP with polynucleotide kinase, as iswell known in the art, to radiolabel the oligonucleotide. However, othermethods may be used to label the oligonucleotide, including, but notlimited to, biotinylation or enzyme labeling.

Amino acid sequence variants of WSX receptor or OB protein are preparedby introducing appropriate nucleotide changes into the WSX receptor orOB protein DNA, or by synthesis of the desired WSX receptor or OBprotein. Such variants represent insertions, substitutions, and/orspecified deletions of, residues within or at one or both of the ends ofthe amino acid sequence of a naturally occurring human WSX receptor orOB protein, such as the WSX receptor variants shown in FIGS. 2A-D or thehuman OB protein of Zhang et al., supra. Preferably, these variantsrepresent insertions and/or substitutions within or at one or both endsof the mature sequence, and/or insertions, substitutions and/orspecificed deletions within or at one or both of the ends of the signalsequence of the WSX receptor or OB protein. Any combination ofinsertion, substitution, and/or specified deletion is made to arrive atthe final construct, provided that the final construct possesses thedesired biological activity as defined herein. The amino acid changesalso may alter post-translational processes of the WSX receptor or OBprotein, such as changing the number or position of glycosylation sites,altering the membrane anchoring characteristics, and/or altering theintracellular location of the WSX receptor or OB protein by inserting,deleting, or otherwise affecting the leader sequence of the WSX receptoror OB protein.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. See also, for example, Table I thereinand the discussion surrounding this table for guidance on selectingamino acids to change, add, or delete.

2. Insertion of Nucleic Acid into Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the WSX receptoror OB protein is inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

a. Signal Sequence Component

The WSX receptor or OB proteins of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. In general, the signal sequence may be acomponent of the vector, or it may be a part of the WSX receptor or OBprotein DNA that is inserted into the vector. The heterologous signalsequence selected preferably is one that is recognized and processed(i.e., cleaved by a signal peptidase) by the host cell. For prokaryotichost cells that do not recognize and process the native WSX receptor orOB protein signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182 issued 23 Apr. 1991), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 362,179published 4 Apr. 1990), or the signal described in WO 90/13646 published15 Nov. 1990. In mammalian cell expression the native signal sequence(e.g., the WSX receptor or OB protein presequence that normally directssecretion of WSX receptor or OB protein from human cells in vivo) issatisfactory, although other mammalian signal sequences may be suitable,such as signal sequences from other animal WSX receptors or OB proteins,and signal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders, for example, the herpessimplex gD signal.

The DNA for such precursor region is ligated in reading frame to DNAencoding the mature WSX receptor or OB protein.

b. Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of WSX receptor or OB protein DNA. However, the recovery ofgenomic DNA encoding WSX receptor or OB protein is more complex thanthat of an exogenously replicated vector because restriction enzymedigestion is required to excise the WSX receptor or OB protein DNA.

c. Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theWSX receptor or OB protein nucleic acid, such as DHFR or thymidinekinase. The mammalian cell transformants are placed under selectionpressure that only the transformants are uniquely adapted to survive byvirtue of having taken up the marker. Selection pressure is imposed byculturing the transformants under conditions in which the concentrationof selection agent in the medium is successively changed, therebyleading to amplification of both the selection gene and the DNA thatencodes WSX receptor or OB protein. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of WSX receptoror OB protein are synthesized from the amplified DNA. Other examples ofamplifiable genes include metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980). The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding WSX receptoror OB protein. This amplification technique can be used with anyotherwise suitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstandingthe presence of endogenous DHFR if, for example, a mutant DHFR gene thatis highly resistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding WSX receptor or OB protein, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics 85:12 (1977). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Bianchi et al.,Curr. Genet. 12:185 (1987). More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for Klactis. Van den Berg, Bio/Technology 8:135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology 9:968-975 (1991).

d. Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the WSXreceptor or OB protein nucleic acid. Promoters are untranslatedsequences located upstream (5′) to the start codon of a structural gene(generally within about 100 to 1000 bp) that control the transcriptionand translation of particular nucleic acid sequence, such as the WSXreceptor or OB protein nucleic acid sequence, to which they are operablylinked. Such promoters typically fall into two classes, inducible andconstitutive. Inducible promoters are promoters that initiate increasedlevels of transcription from DNA under their control in response to somechange in culture conditions, e.g., the presence or absence of anutrient or a change in temperature. At this time a large number ofpromoters recognized by a variety of potential host cells are wellknown. These promoters are operably linked to WSX receptor or OBprotein-encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native WSX receptor or OB proteinpromoter sequence and many heterologous promoters may be used to directamplification and/or expression of the WSX receptor or OB protein DNA.However, heterologous promoters are preferred, as they generally permitgreater transcription and higher yields of WSX receptor or OB protein ascompared to the native WSX receptor or OB protein promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); Goeddel et al., Nature 281:544 (1979)), alkaline phosphatase, atryptophan (trp) promoter system (Goeddel, Nucleic Acids Res. 8:4057(1980); EP 36,776), and hybrid promoters such as the tac promoter.deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983). However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding WSX receptor or OB protein (Siebenlist etal., Cell 20:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding WSX receptor or OB protein.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al., JAdv. Enzyme Reg. 7:149 (1968); Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

WSX receptor or OB protein transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with theWSX receptor or OB protein sequence, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature 273:113 (1978); Mulligan et al.,Science 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA 78:7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene 18:355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani et al.,Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

e. Enhancer Element Component

Transcription of a DNA encoding the WSX receptor or OB protein of thisinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation and positionindependent, having been found 5′ (Laimins et al., Proc. Natl. Acad.Sci. USA 78:993 (1981)) and 3′ (Lusky et al., Mol. Cell Bio. 3:1108(1983)) to the transcription unit, within an intron (Banerji et al.,Cell 33:729 (1983)), as well as within the coding sequence itself.Osborne et al., Mol. Cell Bio. 4:1293 (1984). Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the WSX receptor or OB protein-encoding sequence,but is preferably located at a site 5′ from the promoter.

f. Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding WSX receptor or OB protein.

g. Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology 65:499 (1980).

h. Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding WSX receptor or OB protein. In general, transientexpression involves the use of an expression vector that is able toreplicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Sambrook et al., supra, pp. 16.17-16.22. Transientexpression systems, comprising a suitable expression vector and a hostcell, allow for the convenient positive identification of polypeptidesencoded by cloned DNAs, as well as for the rapid screening of suchpolypeptides for desired biological or physiological properties. Thus,transient expression systems are particularly useful in the inventionfor purposes of identifying analogs and variants of WSX receptor or OBprotein that are biologically active WSX receptor or OB protein.

i. Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of WSX receptor or OB protein in recombinant vertebrate cellculture are described in Gething et al., Nature 293:620-625 (1981);Mantei et al., Nature 281:40-46 (1979); EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression of WSXreceptor or OB protein is pRK5 (EP 307,247) or pSVI6B. WO 91/08291published 13 Jun. 1991.

3. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 is aparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 27C7. The complete genotype of 27C7 is tonAΔ ptr3 phoAΔE15Δ(argF-lac)169 ompTΔ degP41kan^(r). Strain 27C7 was deposited on 30 Oct.1991 in the American Type Culture Collection as ATCC No. 55,244.Alternatively, the strain of E. coli having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 may be employed.Alternatively still, methods of cloning, e.g., PCR or other nucleic acidpolymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for WSX receptoror OB protein-encoding vectors. Saccharomyces cerevisiae, or commonbaker's yeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe (Beach et al., Nature, 290:140 (1981); EP 139,383 published 2 May1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,supra) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis(ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178),K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg etal., supra), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.28:265-278 (1988)); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa (Case et al., Proc. Natl. Acad. Sci. USA 76:5259-5263 (1979));Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun. 112:284-289 (1983); Tilburn et al., Gene26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA81:1470-1474 (1984)) and A. niger. Kelly et al., EMBO J. 4:475-479(1985).

Suitable host cells for the expression of glycosylated WSX receptor orOB protein are derived from multicellular organisms. Such host cells arecapable of complex processing and glycosylation activities. Inprinciple, any higher eukaryotic cell culture is workable, whether fromvertebrate or invertebrate culture. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. See, e.g., Luckow et al., Bio/Technology6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow et al.,eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al.,Nature 315:592-594 (1985). A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the WSX receptor or OB protein-encoding DNA. During incubationof the plant cell culture with A. tumefaciens, the DNA encoding the WSXreceptor or OB protein is transferred to the plant cell host such thatit is transfected, and will, under appropriate conditions, express theWSX receptor or OB protein-encoding DNA. In addition, regulatory andsignal sequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen. 1:561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for WSX receptor or OBprotein production and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene 23:315 (1983) and WO 89/05859published 29 Jun. 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham et al., Virology 52:456-457 (1978) ispreferred. General aspects of mammalian cell host system transformationshave been described in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact. 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. USA 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, etc., may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology 185:527-537 (1990) and Mansour et al., Nature336:348-352 (1988).

4. Culturing the Host Cells

Prokaryotic cells used to produce the WSX receptor or OB protein of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the WSX receptor or OB proteinof this invention may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(DMEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al. Meth. Enz. 58:44(1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430;WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.75:734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared as described herein.

6. Purification of WSX Receptor or OB Protein

WSX receptor (e.g., WSX receptor ECD) or OB protein preferably isrecovered from the culture medium as a secreted polypeptide, although italso may be recovered from host cell lysates. If the WSX receptor ismembrane-bound, it can be released from the membrane using a suitabledetergent solution (e.g. Triton-X 100)

When WSX receptor or OB protein is produced in a recombinant cell otherthan one of human origin, the WSX receptor or OB protein is completelyfree of proteins or polypeptides of human origin. However, it isnecessary to purify WSX receptor or OB protein from recombinant cellproteins or polypeptides to obtain preparations that are substantiallyhomogeneous as to WSX receptor or OB protein. As a first step, theculture medium or lysate is centrifuged to remove particulate celldebris. WSX receptor or OB protein thereafter is purified fromcontaminant soluble proteins and polypeptides, with the followingprocedures being exemplary of suitable purification procedures: byfractionation on an ion-exchange column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75™; and protein A Sepharose™columns to remove contaminants such as IgG.

WSX receptor or OB protein variants in which residues have been deleted,inserted, or substituted are recovered in the same fashion as nativesequence WSX receptor or OB protein, taking account of any substantialchanges in properties occasioned by the variation. Immunoaffinitycolumns such as a rabbit polyclonal anti-WSX receptor or OB proteincolumn can be employed to absorb the WSX receptor or OB protein variantby binding it to at least one remaining immune epitope.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants.

7. Covalent Modifications

Covalent modifications of WSX receptor or OB protein are included withinthe scope of this invention. Both native sequence WSX receptor or OBprotein and amino acid sequence variants of the WSX receptor or OBprotein may be covalently modified. One type of covalent modification ofthe WSX receptor or OB protein is introduced into the molecule byreacting targeted amino acid residues of the WSX receptor or OB proteinwith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the WSXreceptor or OB protein.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para- bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed under alkaline conditionsbecause of the high pK_(a) of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas with the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵1 or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking WSXreceptor or OB protein to a water-insoluble support matrix or surfacefor use in the method for purifying anti-WSX receptor or OB proteinantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the WSX receptor or OB proteinincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. By altering is meantdeleting one or more carbohydrate moieties found in native WSX receptoror OB protein, and/or adding one or more glycosylation sites that arenot present in the native WSX receptor or OB protein.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the WSX receptor or OB protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the native WSX receptor or OB protein sequence (for O-linkedglycosylation sites). For ease, the WSX receptor or OB protein aminoacid sequence is preferably altered through changes at the DNA level,particularly by mutating the DNA encoding the WSX receptor or OB proteinat preselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above and in U.S. Pat. No. 5,364,934, supra.

Another means of increasing the number of carbohydrate moieties on theWSX receptor or OB protein is by chemical or enzymatic coupling ofglycosides to the polypeptide. These procedures are advantageous in thatthey do not require production of the polypeptide in a host cell thathas glycosylation capabilities for N- or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published 11Sep. 1987, and in Aplin et al., CRC Crit. Rev. Biochem. 259-306 (1981).

Removal of carbohydrate moieties present on the WSX receptor or OBprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al., Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al., Anal.Biochem. 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.138:350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem. 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of WSX receptor or OB proteincomprises linking the WSX receptor or OB protein to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Since it is often difficult to predict in advance the characteristics ofa variant WSX receptor or OB protein, it will be appreciated that somescreening of the recovered variant will be needed to select the optimalvariant. A change in the immunological character of the WSX receptor orOB protein molecule, such as affinity for a given antibody, is also ableto be measured by a competitive-type immunoassay. The WSX receptorvariant is assayed for changes in the ability of the protein to inducecell proliferation in the colony assay of Example 8. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

8. Epitope-Tagged WSX Receptor or OB Protein

This invention encompasses chimeric polypeptides comprising WSX receptoror OB protein fused to a heterologous polypeptide. A chimeric WSXreceptor or OB protein is one type of WSX receptor or OB protein variantas defined herein. In one preferred embodiment, the chimeric polypeptidecomprises a fusion of the WSX receptor or OB protein with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally provided at the amino- orcarboxyl-terminus of the WSX receptor or OB protein. Such epitope-taggedforms of the WSX receptor or OB protein are desirable as the presencethereof can be detected using a labeled antibody against the tagpolypeptide. Also, provision of the epitope tag enables the WSX receptoror OB protein to be readily purified by affinity purification using theanti-tag antibody. Affinity purification techniques and diagnosticassays involving antibodies are described later herein.

Tag polypeptides and their respective antibodies are well known in theart. Examples include the flu HA tag polypeptide and its antibody 12CA5(Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,Molecular and Cellular Biology 5:3610-3616 (1985)); and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody. Paborsky et al.,Protein Engineering 3(6):547-553 (1990). Other tag polypeptides havebeen disclosed. Examples include the Flag-peptide (Hopp et al.,BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science 255:192-194 (1992)); an α-tubulin epitope peptide (Skinneret al., J. Biol. Chem. 266:15163-15166 (1991)); and the T7 gene 10protein peptide tag. Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA87:6393-6397 (1990). Once the tag polypeptide has been selected, anantibody thereto can be generated using the techniques disclosed herein.

The general methods suitable for the construction and production ofepitope-tagged WSX receptor or OB protein are the same as thosedisclosed hereinabove. WSX receptor or OB protein-tag polypeptidefusions are most conveniently constructed by fusing the cDNA sequenceencoding the WSX receptor or OB protein portion in-frame to the tagpolypeptide DNA sequence and expressing the resultant DNA fusionconstruct in appropriate host cells. Ordinarily, when preparing the WSXreceptor or OB protein-tag polypeptide chimeras of the presentinvention, nucleic acid encoding the WSX receptor or OB protein will befused at its 3′ end to nucleic acid encoding the N-terminus of the tagpolypeptide, however 5′ fusions are also possible.

Epitope-tagged WSX receptor or OB protein can be conveniently purifiedby affinity chromatography using the anti-tag antibody. The matrix towhich the affinity antibody is attached is most often agarose, but othermatrices are available (e.g. controlled pore glass orpoly(styrenedivinyl)benzene). The epitope-tagged WSX receptor or OBprotein can be eluted from the affinity column by varying the buffer pHor ionic strength or adding chaotropic agents, for example.

9. WSX Receptor or OB Protein Immunoadhesins

Chimeras constructed from a receptor sequence linked to an appropriateimmunoglobulin constant domain sequence (immunoadhesins) are known inthe art. Immunoadhesins reported in the literature include fusions ofthe T cell receptor* (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987)); CD4* (Capon et al., Nature 337: 525-531 (1989);Traunecker et al., Nature 339: 68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9: 347-353 (1990); Byrn et al., Nature 344: 667-670 (1990));L-selectin (homing receptor) ((Watson et al., J. Cell. Biol.110:2221-2229 (1990); Watson et al., Nature 349: 164-167 (1991)); CD44*(Aruffo et al., Cell 61: 1303-1313 (1990)); CD28* and B7* (Linsley etal., J. Exp. Med. 173: 721-730 (1991)); CTLA-4* (Lisley et al., J. Exp.Med. 174: 561-569 (1991)); CD22* (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27: 2883-2886(1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); NP receptors(Bennett et al., J. Biol. Chem. 266:23060-23067 (1991)); and IgEreceptor α* (Ridgway et al., J. Cell. Biol. 115:abstr. 1448 (1991)),where the asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the “adhesin” protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing the WSXreceptor or OB-immunoglobulin chimeras of the present invention, nucleicacid encoding OB protein or the extracellular domain of the WSX receptorwill be fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible. For OB-immunoglobulin chimeras, an OB protein fragmentwhich retains the ability to bind to the WSX receptor may be employed.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the WSXreceptor or OB-immunoglobulin chimeras.

In some embodiments, the WSX receptor or OB-immunoglobulin chimeras areassembled as monomers, or hetero- or homo-multimers, and particularly asdimers or tetramers, essentially as illustrated in WO 91/08298.

In a preferred embodiment, the OB protein sequence or WSX receptorextracellular domain sequence is fused to the N-terminus of theC-terminal portion of an antibody (in particular the Fc domain),containing the effector functions of an immunoglobulin, e.g.immunoglobulin G1 (IgG1). It is possible to fuse the entire heavy chainconstant region to the OB protein or WSX receptor extracellular domainsequence. However, more preferably, a sequence beginning in the hingeregion just upstream of the papain cleavage site (which defines IgG Fcchemically; residue 216, taking the first residue of heavy chainconstant region to be 114, or analogous sites of other immunoglobulins)is used in the fusion. In a particularly preferred embodiment, the OBprotein or WSX receptor amino acid sequence is fused to the hingeregion, CH2 and CH3, or the CH1, hinge, CH2 and CH3 domains of an IgG1,IgG2, or IgG3 heavy chain. The precise site at which the fusion is madeis not critical, and the optimal site can be determined by routineexperimentation.

In some embodiments, the WSX receptor or OB-immunoglobulin chimeras areassembled as multimers, and particularly as homo-dimers or -tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

Various exemplary assembled WSX receptor or OB-immunoglobulin chimeraswithin the scope herein are schematically diagrammed below:

-   -   (a) AC_(L)-AC_(L);    -   (b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H));    -   (c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H));    -   (d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H));    -   (e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H));        and    -   (f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,    -   wherein    -   each A represents identical or different OB protein or WSX        receptor amino acid sequences;    -   V_(L) is an immunoglobulin light chain variable domain;    -   V_(H) is an immunoglobulin heavy chain variable domain;    -   C_(L) is an immunoglobulin light chain constant domain;    -   C_(H) is an immunoglobulin heavy chain constant domain;    -   n is an integer greater than 1;    -   Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the OB protein or WSX receptor extracellular domainsequence can be inserted between immunoglobulin heavy chain and lightchain sequences such that an immunoglobulin comprising a chimeric heavychain is obtained. In this embodiment, the OB protein or WSX receptorsequence is fused to the 3′ end of an immunoglobulin heavy chain in eacharm of an immunoglobulin, either between the hinge and the CH2 domain,or between the CH2 and CH3 domains. Similar constructs have beenreported by Hoogenboom et al, Mol. Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to an OB protein orWSX receptor-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the WSX receptor extracellular domain or OB protein. In theformer case, DNA encoding an immunoglobulin light chain is typicallycoexpressed with the DNA encoding the OB protein or WSXreceptor-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567 issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG1 and IgG3 immunoglobulin sequencesis preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger adhesindomains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantly weaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in vivo half-life is approximately one third of the other IgGisotypes. Another important consideration for immunoadhesins designed tobe used as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG1 hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3 m5, 11 and 21) have one allotype which isnonimmunogenic. Thus, the potential immunogenicity of a γ3 immunoadhesinis greater than that of a γ1 immunoadhesin.

With respect to the parental immunoglobulin, a useful joining point isjust upstream of the cysteines of the hinge that form the disulfidebonds between the two heavy chains. In a frequently used design, thecodon for the C-terminal residue of the WSX receptor or OB protein partof the molecule is placed directly upstream of the codons for thesequence DKTHTCPPCP (SEQ ID NO:44) of the IgG1 hinge region.

The general methods suitable for the construction and expression ofimmunoadhesins are the same as those disclosed hereinabove with regardto WSX receptor and OB protein. Immunoadhesins are most convenientlyconstructed by fusing the cDNA sequence encoding the WSX receptor or OBprotein portion in-frame to an Ig cDNA sequence. However, fusion togenomic Ig fragments can also be used (see, e.g., Gascoigne et al.,Proc. Natl. Acad. Sci. USA, 84:2936-2940 (1987); Aruffo et al., Cell61:1303-1313 (1990); Stamenkovic et al., Cell 66:1133-1144 (1991)). Thelatter type of fusion requires the presence of Ig regulatory sequencesfor expression. cDNAs encoding IgG heavy-chain constant regions can beisolated based on published sequence from cDNA libraries derived fromspleen or peripheral blood lymphocytes, by hybridization or bypolymerase chain reaction (PCR) techniques. The cDNAs encoding the WSXreceptor or OB protein and Ig parts of the immunoadhesin are inserted intandem into a plasmid vector that directs efficient expression in thechosen host cells. For expression in mammalian cells, pRK5-based vectors(Schall et al., Cell 61:361-370 (1990)) and CDM8-based vectors (Seed,Nature 329:840 (1989)) can be used. The exact junction can be created byremoving the extra sequences between the designed junction codons usingoligonucleotide-directed deletional mutagenesis (Zoller et al., NucleicAcids Res. 10:6487 (1982); Capon et al., Nature 337:525-531 (1989)).Synthetic oligonucleotides can be used, in which each half iscomplementary to the sequence on either side of the desired junction;ideally, these are 36 to 48-mers. Alternatively, PCR techniques can beused to join the two parts of the molecule in-frame with an appropriatevector.

The choice of host cell line for the expression of the immunoadhesindepends mainly on the expression vector. Another consideration is theamount of protein that is required. Milligram quantities often can beproduced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5-based vectors by a modification of the calciumphosphate method to allow efficient immunoadhesin expression. CDM8-basedvectors can be used to transfect COS cells by the DEAE-dextran method(Aruffo et al., Cell 61:1303-1313 (1990); Zettmeissl et al., DNA CellBiol. US 9:347-353 (1990)). If larger amounts of protein are desired,the immunoadhesin can be expressed after stable transfection of a hostcell line. For example, a pRK5-based vector can be introduced intoChinese hamster ovary (CHO) cells in the presence of an additionalplasmid encoding dihydrofolate reductase (DHFR) and conferringresistance to G418. Clones resistant to G418 can be selected in culture;these clones are grown in the presence of increasing levels of DHFRinhibitor methotrexate; clones are selected, in which the number of genecopies encoding the DHFR and immunoadhesin sequences is co-amplified. Ifthe immunoadhesin contains a hydrophobic leader sequence at itsN-terminus, it is likely to be processed and secreted by the transfectedcells. The expression of immunoadhesins with more complex structures mayrequire uniquely suited host cells; for example, components such aslight chain or J chain may be provided by certain myeloma or hybridomacell hosts (Gascoigne et al., 1987, supra, Martin et al., J. Virol.67:3561-3568 (1993)).

Immunoadhesins can be conveniently purified by affinity chromatography.The suitability of protein A as an affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Theconditions for binding an immunoadhesin to the protein A or G affinitycolumn are dictated entirely by the characteristics of the Fc domain;that is, its species and isotype. Generally, when the proper ligand ischosen, efficient binding occurs directly from unconditioned culturefluid. One distinguishing feature of immunoadhesins is that, for humanγ1 molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in an immunoadhesin preparation thatis >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatography on protein A or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodies in thiophilic gelchromatography (Hutchens et al., Anal. Biochem. 159:217-226 (1986)) andimmobilized metal chelate chromatography (Al-Mashikhi et al., J. DairySci. 71:1756-1763 (1988)). In contrast to antibodies, however, theirbehavior on ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

If desired, the immunoadhesins can be made bispecific. Thus, theimmunoadhesins of the present invention may combine a WSX receptorextracellular domain and a domain, such as the extracellular domain, ofanother cytokine receptor subunit. Exemplary cytokine receptors fromwhich such bispecific immunoadhesin molecules can be made include TPO(or mpl ligand), EPO, G-CSF, IL-4, IL-7, GH, PRL, IL-3, GM-CSF, IL-5,IL-6, LIF, OSM, CNTF and IL-2 receptors. Alternatively, an OB proteindomain may be combined with another cytokine, such as those exemplifiedherein, in the generation of a bispecific immunoadhesin. For bispecificmolecules, trimeric molecules, composed of a chimeric antibody heavychain in one arm and a chimeric antibody heavy chain-light chain pair inthe other arm of their antibody-like structure are advantageous, due toease of purification. In contrast to antibody-producing quadromastraditionally used for the production of bispecific immunoadhesins,which produce a mixture of ten tetramers, cells transfected with nucleicacid encoding the three chains of a trimeric immunoadhesin structureproduce a mixture of only three molecules, and purification of thedesired product from this mixture is correspondingly easier.

10. Long Half-Life Derivatives of OB Protein

Prefered OB protein functional derivatives for use in the methods of thepresent invention include OB-immunoglobulin chimeras (immunoadhesins)and other longer half-life molecules. Techniques for generating OBprotein immunoadhesins have been described above. The prefered OBimmunoadhesin is made according to the techniques described in Example11 below.

Other derivatives of the OB proteins, which possess a longer half-lifethan the native molecules comprise the OB protein or anOB-immunoglobulin chimera covalently bonded to a nonproteinaceouspolymer. The nonproteinaceous polymer ordinarily is a hydrophilicsynthetic polymer, i.e., a polymer not otherwise found in nature.However, polymers which exist in nature and are produced by recombinantor in vitro methods are useful, as are polymers which are isolated fromnative sources. Hydrophilic polyvinyl polymers fall within the scope ofthis invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.Particularly useful are polyalkylene ethers such as polyethylene glycol(PEG); polyelkylenes such as polyoxyethylene, polyoxypropylene, andblock copolymers of polyoxyethylene and polyoxypropylene (Pluronics™);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextranesulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparon. Thepolymer prior to cross-linking need not be, but preferably is, watersoluble, but the final conjugate must be water soluble. In addition, thepolymer should not be highly immunogenic in the conjugate form, norshould it possess viscosity that is incompatible with intravenousinfusion or injection if it is intended to be administered by suchroutes.

Preferably the polymer contains only a single group which is reactive.This helps to avoid cross-linking of protein molecules. However, it iswithin the scope herein to optimize reaction conditions to reducecross-linking, or to purify the reaction products through gel filtrationor chromatographic sieves to recover substantially homogenousderivatives.

The molecular weight of the polymer may desirably range from about 100to 500,000, and preferably is from about 1,000 to 20,000. The molecularweight chosen will depend upon the nature of the polymer and the degreeof substitution. In general, the greater the hydrophilicity of thepolymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

The polymer generally is covalently linked to the OB protein or to theOB-immunoglobulin chimera though a multifunctional crosslinking agentwhich reacts with the polymer and one or more amino acid or sugarresidues of the OB protein or OB-immunoglobulin chimera to be linked.However, it is within the scope of the invention to directly crosslinkthe polymer by reacting a derivatized polymer with the hybrid, or viaversa.

The covalent crosslinking site on the OB protein or OB-immunoglobulinchimera includes the N-terminal amino group and epsilon amino groupsfound on lysine residues, as well as other amino, imino, carboxyl,sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may becovalently bonded directly to the hybrid without the use of amultifunctional (ordinarily bifunctional) crosslinking agent. Covalentbinding to amino groups is accomplished by known chemistries based uponcyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEGalkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO andacetic anhydride, or PEG chloride plus the phenoxide of4-hydroxybenzaldehyde, succinimidyl active esters, activateddithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate orP-nitrophenylcloroformate activated PEG). Carboxyl groups arederivatized by coupling PEG-amine using carbodiimide.

Polymers are conjugated to oligosaccharide groups by oxidation usingchemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactoseoxidase (either of which produces the aldehyde derivative of thecarbohydrate), followed by reaction with hydrazide or amino derivatizedpolymers, in the same fashion as is described by Heitzmann et al.,P.N.A.S. 71:3537-41 (1974) or Bayer et al., Methods in Enzymology 62:310(1979), for the labeling of oligosaccharides with biotin or avidin.Further, other chemical or enzymatic methods which have been usedheretofore to link oligosaccharides are particularly advantageousbecause, in general, there are fewer substitutions than amino acid sitesfor derivatization, and the oligosaccharide products thus will be morehomogenous. The oligosaccharide substituents also are optionallymodified by enzyme digestion to remove sugars, e.g. by neuraminidasedigestion, prior to polymer derivatization.

The polymer will bear a group which is directly reactive with an aminoacid side chain, or the N- or C-terminus of the polypeptide linked, orwhich is reactive with the multifunctional cross-linking agent. Ingeneral, polymers bearing such reactive groups are known for thepreparation of immobilized proteins. In order to use such chemistrieshere, one should employ a water soluble polymer otherwise derivatized inthe same fashion as insoluble polymers heretofore employed for proteinimmobilization. Cyanogen bromide activation is a particularly usefulprocedure to employ in crosslinking polysaccharides.

“Water soluble” in reference to the starting polymer means that thepolymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.

“Water soluble” in reference to the polymer conjugate means that theconjugate is soluble in physiological fluids such as blood.

The degree of substitution with such a polymer will vary depending uponthe number of reactive sites on the protein, whether all or a fragmentof the protein is used, whether the protein is a fusion with aheterologous protein (e.g. an OB-immunoglobulin chimera), the molecularweight, hydrophilicity and other characteristics of the polymer, and theparticular protein derivatization sites chosen. In general, theconjugate contains about from 1 to 10 polymer molecules, while anyheterologous sequence may be substituted with an essentially unlimitednumber of polymer molecules so long as the desired activity is notsignificantly adversely affected. The optimal degree of cross-linking iseasily determined by an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the ability of the conjugates tofunction in the desired fashion is determined.

The polymer, e.g. PEG, is cross-linked by a wide variety of methodsknown per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG. Certain of these methods,however, are not preferred for the purposes herein. Cyanuronic chloridechemistry leads to many side reactions, including protein cross-linking.In addition, it may be particularly likely to lead to inactivation ofproteins containing sulfhydryl groups. Carbonyl diimidazole chemistry(Beauchamp et al., Anal Biochem. 131:25-33 (1983)) requires high pH(>8.5), which can inactivate proteins. Moreover, since the “activatedPEG” intermediate can react with water, a very large molar excess of“activated PEG” over protein is required. The high concentrations of PEGrequired for the carbonyl diimidazole chemistry also led to problems inpurification, as both gel filtration chromatography and hydrophilicinteraction chromatography are adversely affected. In addition, the highconcentrations of “activated PEG” may precipitate protein, a problemthat per se has been noted previously (Davis, U.S. Pat. No. 4,179,337).On the other hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531)is more efficient since it requires only a 40-fold molar excess of PEGand a 1-2 hr incubation. However, the manganese dioxide suggested byRoyer for preparation of the PEG aldehyde is problematic “because of thepronounced tendency of PEG to form complexes with metal-based oxidizingagents” (Harris et al., J Polym. Sci. Polym. Chem. Ed. 22:341-52(1984)). The use of a Moffatt oxidation, utilizing DMSO and aceticanhydride, obviates this problem. In addition, the sodium borohydridesuggested by Royer must be used at high pH and has a significanttendency to reduce disulfide bonds. In contrast, sodiumcyanoborohydride, which is effective at neutral pH and has very littletendency to reduce disulfide bonds is preferred.

Functionalized PEG polymers to modify the OB protein orOB-immunoglobulin chimeras of the present invention are available fromShearwater Polymers, Inc. (Huntsville, Ala.). Such commerciallyavailable PEG derivatives include, but are not limited to, amino-PEG,PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate,carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEGsuccinimidyl succinate, PEG succinimidyl propionate, succinimidyl esterof carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidylesters of amino acid PEGs, PEG-oxycarbonylimidazole, PEG-nitrophenylcarbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEGvinylsulfone, PEG-maleimide, PEG-orthopyridyl-disulfide,heterofunctional PEGs, PEG vinyl derivatives, PEG silanes, and PEGphospholides. The reaction conditions for coupling these PEG derivativeswill vary depending on the protein, the desired degree of PEGylation,and the PEG derivative utilized. Some factors involved in the choice ofPEG derivatives include: the desired point of attachment (lysine orcysteine), hydrolytic stability and reactivity of the derivatives,stability, toxicity and antigenicity of the linkage, suitability foranalysis, etc. Specific instructions for the use of any particularderivative are available from the manufacturer.

The long half-life conjugates of this invention are separated from theunreacted starting materials by gel filtration. Heterologous species ofthe conjugates are purified from one another in the same fashion. Thepolymer also may be water-insoluble, as a hydrophilic gel.

The conjugates may also be purified by ion-exchange chromatography. Thechemistry of many of the electrophilically activated PEG's results in areduction of amino group charge of the PEGylated product. Thus, highresolution ion exchange chromatography can be used to separate the freeand conjugated proteins, and to resolve species with different levels ofPEGylation. In fact, the resolution of different species (e.g.containing one or two PEG residues) is also possible due to thedifference in the ionic properties of the unreacted amino acids.

B. Therapeutic Uses for the WSX Receptor

The WSX receptor and WSX receptor gene are believed to find therapeuticuse for administration to a mammal in the treatment of diseasescharacterized by a decrease in hematopoietic cells. Examples of thesediseases include: anemia (including macrocytic and aplastic anemia);thrombocytopenia; hypoplasia; disseminated intravascular coagulation(DIC); myelodysplasia; immune (autoimmune) thrombocytopenic purpura(ITP); and HIV induced ITP. Additionally, these WSX receptor moleculesmay be useful in treating myeloproliferative thrombocytotic diseases aswell as thrombocytosis from inflammatory conditions and in irondeficiency. WSX receptor polypeptide and WSX receptor gene which lead toan increase in hematopoietic cell proliferation may also be used toenhance repopulation of mature blood cell lineages in cells havingundergone chemo- or radiation therapy or bone marrow transplantationtherapy. Generally, the WSX receptor molecules are expected to lead toan enhancement of the proliferation and/or differentiation (butespecially proliferation) of primitive hematopoietic cells. Otherpotential therapeutic applications for WSX receptor and WSX receptorgene include the treatment of obesity and diabetes and for promotingkidney, liver and lung growth and/or repair (e.g. in renal failure).

The WSX receptor may be administered alone or in combination withcytokines (such as OB protein), growth factors or antibodies in theabove-identified clinical situations. This may facilitate an effectivelowering of the dose of WSX receptor. Suitable dosages for suchadditional molecules will be discussed below.

Administration of WSX receptor to a mammal having depressed levels ofendogenous WSX receptor or a defective WSX receptor gene iscontemplated, preferably in the situation where such depressed levelslead to a pathological disorder, or where there is lack of activation ofthe WSX receptor. In these embodiments where the full length WSXreceptor is to be administered to the patient, it is contemplated thatthe gene encoding the receptor may be administered to the patient viagene therapy technology.

In gene therapy applications, genes are introduced into cells in orderto achieve in vivo synthesis of a therapeutically effective geneticproduct, for example for replacement of a defective gene. “Gene therapy”includes both conventional gene therapy where a lasting effect isachieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA, 83:4143-4146 (1986)). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993)).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992).

The invention also provides antagonists of WSX receptor activation (e.g.WSX receptor ECD, WSX receptor immunoadhesins and WSX receptor antisensenucleic acid; neutralizing antibodies and uses thereof are discussed insection E below). Administration of WSX receptor antagonist to a mammalhaving increased or excessive levels of endogenous WSX receptoractivation is contemplated, preferably in the situation where suchlevels of WSX receptor activation lead to a pathological disorder.

In one embodiment, WSX receptor antagonist molecules may be used to bindendogenous ligand in the body, thereby causing desensitized WSXreceptors to become responsive to WSX ligand, especially when the levelsof WSX ligand in the serum exceed normal physiological levels. Also, itmay be beneficial to bind endogenous WSX ligand which is activatingundesired cellular responses (such as proliferation of tumor cells).Potential therapeutic applications for WSX antagonists include forexample, treatment of metabolic disorders (e.g., anorexia andsteroid-induced truncalobesity), stem cell tumors and other tumors whichexpress WSX receptor.

Pharmaceutical compositions of the WSX receptor ECD may further includea WSX ligand. Such dual compositions may be beneficial where it istherapeutically useful to prolong half-life of WSX ligand, and/oractivate endogenous WSX receptor directly as a heterotrimeric complex.

Therapeutic formulations of WSX receptor are prepared for storage bymixing WSX receptor having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter-ions such as sodium; and/or non-ionic surfactantssuch as Tween, Pluronics™ or polyethylene glycol (PEG).

The WSX receptor also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

WSX receptor to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. WSXreceptor ordinarily will be stored in lyophilized form or in solution.

Therapeutic WSX receptor compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

The route of WSX receptor administration is in accord with knownmethods, e.g., those routes set forth above for specific indications, aswell as the general routes of injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional means, or sustained release systems asnoted below. WSX receptor is administered continuously by infusion or bybolus injection. Generally, where the disorder permits, one shouldformulate and dose the WSX receptor for site-specific delivery.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981) andLanger, Chem. Tech. 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and γ ethyl-L-glutamate (Sidman et al., Biopolymers22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release WSX receptor compositions also include liposomallyentrapped WSX receptor. Liposomes containing WSX receptor are preparedby methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad.Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal WSX receptor therapy.

When applied topically, the WSX receptor is suitably combined with otheringredients, such as carriers and/or adjuvants. There are no limitationson the nature of such other ingredients, except that they must bephysiologically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the WSX receptor formulated in a liquidcomposition may be mixed with an effective amount of a water-solublepolysaccharide or synthetic polymer such as PEG to form a gel of theproper viscosity to be applied topically. The polysaccharide that may beused includes, for example, cellulose derivatives such as etherifiedcellulose derivatives, including alkyl celluloses, hydroxyalkylcelluloses, and alkylhydroxyalkyl celluloses, for example,methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch andfractionated starch; agar; alginic acid and alginates; gum arabic;pullullan; agarose; carrageenan; dextrans; dextrins; fructans; inulin;mannans; xylans; arabinans; chitosans; glycogens; glucans; and syntheticbiopolymers; as well as gums such as xanthan gum; guar gum; locust beangum; gum arabic; tragacanth gum; and karaya gum; and derivatives andmixtures thereof. The preferred gelling agent herein is one that isinert to biological systems, nontoxic, simple to prepare, and not toorunny or viscous, and will not destabilize the WSX receptor held withinit.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight PEGs to obtain the proper viscosity. Forexample, a mixture of a PEG of molecular weight 400-600 with one ofmolecular weight 1500 would be effective for this purpose when mixed inthe proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides and PEGs ismeant to include colloidal solutions and dispersions. In general, thesolubility of the cellulose derivatives is determined by the degree ofsubstitution of ether groups, and the stabilizing derivatives usefulherein should have a sufficient quantity of such ether groups peranhydroglucose unit in the cellulose chain to render the derivativeswater soluble. A degree of ether substitution of at least 0.35 ethergroups per anhydroglucose unit is generally sufficient. Additionally,the cellulose derivatives may be in the form of alkali metal salts, forexample, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2- 5%, more preferably about 3%, of the gel and the WSX receptor ispresent in an amount of about 300-1000 mg per ml of gel.

An effective amount of WSX receptor to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the WSX receptor until a dosageis reached that achieves the desired effect. A typical daily dosage forsystemic treatment might range from about 1 μg/kg to up to 10 mg/kg ormore, depending on the factors mentioned above. As an alternativegeneral proposition, the WSX receptor is formulated and delivered to thetarget site or tissue at a dosage capable of establishing in the tissuea WSX receptor level greater than about 0.1 ng/cc up to a maximum dosethat is efficacious but not unduly toxic. This intra-tissueconcentration should be maintained if possible by continuous infusion,sustained release, topical application, or injection at empiricallydetermined frequencies. The progress of this therapy is easily monitoredby conventional assays.

C. Non-Therapeutic Uses for the WSX Receptor

WSX receptor nucleic acid is useful for the preparation of WSX receptorpolypeptide by recombinant techniques exemplified herein which can thenbe used for production of anti-WSX receptor antibodies having variousutilities described below.

The WSX receptor (polypeptide or nucleic acid) can be used to induceproliferation and/or differentiation of cells in vitro. In particular,it is contemplated that this molecule may be used to induceproliferation of stem cell/progenitor cell populations (e.g. CD34+ cellpopulations obtained as described in Example 8 below). These cells whichare to be grown ex vivo may simultaneously be exposed to other knowngrowth factors or cytokines, such as those described herein. Thisresults in proliferation and/or differentiation of the cells having theWSX receptor.

In yet another aspect of the invention, the WSX receptor may be used foraffinity purification of WSX ligand. Briefly, this technique involves:(a) contacting a source of WSX ligand with an immobilized WSX receptorunder conditions whereby the WSX ligand to be purified is selectivelyadsorbed onto the immobilized receptor; (b) washing the immobilized WSXreceptor and its support to remove non-adsorbed material; and (c)eluting the WSX ligand molecules from the immobilized WSX receptor towhich they are adsorbed with an elution buffer. In a particularlypreferred embodiment of affinity purification, WSX receptor iscovalently attaching to an inert and porous matrix (e.g., agarosereacted with cyanogen bromide). Especially preferred is a WSX receptorimmunoadhesin immobilized on a protein A column. A solution containingWSX ligand is then passed through the chromatographic material. The WSXligand adsorbs to the column and is subsequently released by changingthe elution conditions (e.g. by changing pH or ionic strength).

The WSX receptor may be used for competitive screening of potentialagonists or antagonists for binding to the WSX receptor. Such agonistsor antagonists may constitute potential therapeutics for treatingconditions characterized by insufficient or excessive WSX receptoractivation, respectively.

The preferred technique for identifying molecules which bind to the WSXreceptor utilizes a chimeric receptor (e.g., epitope tagged WSX receptoror WSX receptor immunoadhesin) attached to a solid phase, such as thewell of an assay plate. Binding of molecules which are optionallylabelled (e.g., radiolabelled) to the immobilized receptor can beevaluated.

To identify WSX receptor agonists or antagonists, the thymidineincorporation assay can be used. For screening for antagonists, the WSXreceptor can be exposed to a WSX ligand followed by the putativeantagonist, or the WSX ligand and antagonist can be added to the WSXreceptor simultaneously, and the ability of the antagonist to blockreceptor activation can be evaluated.

The WSX receptor polypeptides are also useful as molecular weightmarkers. To use a WSX receptor polypeptide as a molecular weight marker,gel filtration chromatography or SDS-PAGE, for example, will be used toseparate protein(s) for which it is desired to determine their molecularweight(s) in substantially the normal way. The WSX receptor and othermolecular weight markers will be used as standards to provide a range ofmolecular weights. For example, phosphorylase b (mw=97,400), bovineserum albumin (mw=68,000), ovalbumin (mw=46,000), WSX receptor(mw=44,800), trypsin inhibitor (mw=20,100), and lysozyme (mw=14,400) canbe used as mw markers. The other molecular weight markers mentioned herecan be purchased commercially from Amersham Corporation, ArlingtonHeights, Ill. The molecular weight markers are generally labeled tofacilitate detection thereof. For example, the markers may bebiotinylated and following separation can be incubated withstreptavidin-horseradish peroxidase so that the various markers can bedetected by light detection.

The purified WSX receptor, and the nucleic acid encoding it, may also besold as reagents for mechanism studies of WSX receptor and its ligands,to study the role of the WSX receptor and WSX ligand in normal growthand development, as well as abnormal growth and development, e.g., inmalignancies.

WSX receptor variants are useful as standards or controls in assays forthe WSX receptor for example ELISA, RIA, or RRA, provided that they arerecognized by the analytical system employed, e.g., an anti-WSX receptorantibody.

D. WSX Receptor Antibody Preparation

1. Polyclonal Antibodies

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. In that the preferred epitope is in the ECD ofthe WSX receptor, it is desirable to use WSX receptor ECD or a moleculecomprising the ECD (e.g., WSX receptor immunoadhesin) as the antigen forgeneration of polyclonal and monoclonal antibodies. It may be useful toconjugate the relevant antigen to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining 1 mg or 1 μg of the peptide or conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later the animals are boosted with ⅕ to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later the animals are bledand the serum is assayed for antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal is boosted with the conjugateof the same antigen, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature 256:495 (1975), or maybe made by recombinant DNA methods (Cabilly et al., supra).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA encoding themonoclonal antibodies is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of murine antibodies). The hybridoma cells serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, Chinese hamster ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature 348:552-554 (1990). Clackson etal., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Mark et al., Bio/Technology 10:779-783 (1992)), as wellas combinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Cabilly et al., supra; Morrison, etal., Proc. Nat. Acad. Sci. USA 81:6851 (1984)), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Typically such non-immunoglobulinpolypeptides are substituted for the constant domains of an antibody, orthey are substituted for the variable domains of one antigen-combiningsite of an antibody to create a chimeric bivalent antibody comprisingone antigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

3. Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovitset al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno.7:33 (1993). Human antibodies can also be produced in phage-displaylibraries (Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Marks etal., J. Mol. Biol. 222:581 (1991)).

4. Bispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different antigens. BsAbs can be used astumor targeting or imaging agents and can be used to target enzymes ortoxins to a cell possessing the WSX receptor. Such antibodies can bederived from full length antibodies or antibody fragments (e.g. F(ab′)₂bispecific antibodies). In accordance with the present invention, theBsAb may possess one arm which binds the WSX receptor and another armwhich binds to a cytokine or another cytokine receptor (or a subunitthereof) such as the receptors for TPO, EPO, G-CSF, IL-4, IL-7, GH, PRL;the α or β subunits of the IL-3, GM-CSF, IL-5, IL-6, LIF, OSM and CNTFreceptors; or the α, β or γ subunits of the IL-2 receptor complex. Forexample, the BsAb may bind both WSX receptor and gp130.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, published 13 May 1993, and inTraunecker et al., EMBO J. 10:3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. According to these techniques, Fab′-SHfragments can be recovered from E. coli, which can be chemically coupledto form bivalent antibodies. Shalaby et al., J. Exp. Med. 175:217-225(1992) describe the production of a fully humanized BsAb F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the BsAb. TheBsAb thus formed was able to bind to cells overexpressing the HER2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets. Seealso Rodrigues et al., Int. J. Cancers (Suppl.) 7:45-50 (1992).

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making BsAb fragments. Thefragments comprise a heavy-chain variable domain (V_(H)) connected to alight-chain variable domain (V_(L)) by a linker which is too short toallow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for making BsAbfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol. 152:5368 (1994).

E. Therapeutic Uses for WSX Receptor Ligands and Antibodies

The WSX ligands (e.g. OB protein and anti-WSX receptor agonistantibodies) of the present invention are useful, in one embodiment, forweight reduction, and specifically, in the treatment of obesity andother disorders associated with the abnormal expression or function ofthe OB gene, other metabolic disorders such as diabetes and bulimia, forreducing excessive levels of insulin in human patients (e.g. to restoreor improve the insulin-sensitivity of such patients).

In addition, the WSX ligands can be used for the treatment of kidneyailments, hypertension, and lung dysfunctions, such as emphysema.

In a further embodiment, the WSX ligands (such as agonist WSX receptorantibodies) of the present invention can be used to enhance repopulationof mature blood cell lineages in mammals having undergone chemo- orradiation therapy or bone marrow transplantation therapy. Generally, theligands will act via an enhancement of the proliferation and/ordifferentiation (but especially proliferation) of primitivehematopoietic cells. The ligands may similarly be useful for treatingdiseases characterized by a decrease in blood cells. Examples of thesediseases include: anemia (including macrocytic and aplastic anemia);thrombocytopenia; hypoplasia; immune (autoimmune) thrombocytopenicpurpura (ITP); and HIV induced ITP. Also, the ligands may be used totreat a patient having suffered a hemorrhage. WSX ligands may also beused to treat metabolic disorders such as obesity and diabetes mellitus,or to promote kidney, liver or lung growth and/or repair (e.g., in renalfailure).

The WSX receptor ligands and antibodies may be administered alone or inconcert with one or more cytokines. Furthermore, as an alternative toadminstration of the WSX ligand protein, gene therapy techniques(discussed in the section above entitled “Therapeutic Uses for the WSXReceptor”) are also contemplated herein.

Potential therapeutic applications for WSX receptor neutralizingantibodies include the treatment of metabolic disorders (such ascachexia, anorexia and bulimia), stem cell tumors and other tumors atsites of WSX receptor expression, especially those tumors characterizedby overexpression of WSX receptor.

For therapeutic applications, the WSX receptor ligands and antibodies ofthe invention are administered to a mammal, preferably a human, in aphysiologically acceptable dosage form, including those that may beadministered to a human intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intra-cerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The WSX receptorligands and antibodies also are suitably administered by intratumoral,peritumoral, intralesional, or perilesional routes or to the lymph, toexert local as well as systemic therapeutic effects.

Such dosage forms encompass physiologically acceptable carriers that areinherently non-toxic and non-therapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms of WSXreceptor antibodies include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, andwood wax alcohols. For all administrations, conventional depot forms aresuitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The WSX receptorligand or antibody will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing the WSXreceptor ligand or antibody, which matrices are in the form of shapedarticles, e.g. films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., supraand Langer, supra, or poly(vinylalcohol), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate(Sidman et al., supra), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated WSX receptor antibodies remain in the body for a long time,they may denature or aggregate as a result of exposure to moisture at37° C., resulting in a loss of biological activity and possible changesin immunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Sustained-release WSX receptor ligand or antibody compositions alsoinclude liposomally entrapped antibodies. Liposomes containing the WSXreceptor ligand or antibody are prepared by methods known in the art,such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Ordinarily, the liposomes arethe small (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal WSX receptor ligand orantibody therapy. Liposomes with enhanced circulation time are disclosedin U.S. Pat. No. 5,013,556.

For the prevention or treatment of disease, the appropriate dosage ofWSX receptor ligand or antibody will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the antibodies are administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the WSX receptor ligand or antibody, and the discretion of theattending physician. The WSX receptor ligand or antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg of WSX receptor ligand or antibody is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 μg/kg (e.g. 1-50 μg/kg) ormore, depending on the factors mentioned above. For example, the dosemay be the same as that for other cytokines such as G-CSF, GM-CSF andEPO. For repeated administrations over several days or longer, dependingon the condition, the treatment is sustained until a desired suppressionof disease symptoms occurs. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

When one or more cytokines are co-administered with the WSX receptorligand, lesser doses of the WSX ligand may be employed. Suitable dosesof a cytokine are from about 1 g/kg to about 15 mg/kg of cytokine. Atypical daily dosage of the cytokine might range from about 1 μg/kg to100 μg/kg (e.g. 1-50 μg/kg) or more. For example, the dose may be thesame as that for other cytokines such as G-CSF, GM-CSF and EPO. Thecytokine(s) may be administered prior to, simultaneously with, orfollowing administration of the WSX ligand. The cytokine(s) and WSXligand may be combined to form a pharmaceutically composition forsimultaneous administration to the mammal. In certain embodiments, theamounts of WSX ligand and cytokine are such that a synergisticrepopulation of blood cells (or synergistic increase in proliferationand/or differentiation of hematopoietic cells) occurs in the mammal uponadministration of the WSX ligand and cytokine thereto. In other words,the coordinated action of the two or more agents (i.e. the WSX ligandand cytokine(s)) with respect to repopulation of blood cells (orproliferation/differentiation of hematopoietic cells) is greater thanthe sum of the individual effects of these molecules.

F. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the conditionsdescribed above is provided. The article of manufacture comprises acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for treating the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is theWSX ligand. The label on, or associated with, the container indicatesthat the composition is used for treating the condition of choice. Thearticle of manufacture may further comprise a second container holding acytokine for co-administration with the WSX ligand. Further container(s)may be provided with the article of manufacture which may hold, forexample, a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution or dextrose solution. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

G. Non-Therapeutic Uses for WSX Receptor Ligands and Antibodies

WSX receptor ligands and antibodies may be used for detection of and/orenrichment of hematopoietic stem cell/progenitor cell populations in asimilar manner to that in which CD34 antibodies are presently used. Forstem cell enrichment, the WSX receptor antibodies may be utilized in thetechniques known in the art such as immune panning, flow cytometry orimmunomagnetic beads.

In accordance with one in vitro application of the WSX ligands, cellscomprising the WSX receptor are provided and placed in a cell culturemedium. Examples of such WSX-receptor-containing cells includehematopoietic progenitor cells, such as CD34+ cells.

Suitable tissue culture media are well known to persons skilled in theart and include, but are not limited to, Minimal Essential Medium (MEM),RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). These tissueculture medias are commercially available from Sigma Chemical Company(St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells are thencultured in the cell culture medium under conditions sufficient for thecells to remain viable and grow in the presence of an effective amountof WSX ligand and, optionally, further cytokines and growth factors. Thecells can be cultured in a variety of ways, including culturing in aclot, agar, or liquid culture.

The cells are cultured at a physiologically acceptable temperature suchas 37° C., for example, in the presence of an effective amount of WSXligand. The amount of WSX ligand may vary, but preferably is in therange of about 10 ng/ml to about 1 mg/ml. The WSX ligand can of coursebe added to the culture at a dose determined empirically by those in theart without undue experimentation. The concentration of WSX ligand inthe culture will depend on various factors, such as the conditions underwhich the cells and WSX ligand are cultured. The specific temperatureand duration of incubation, as well as other culture conditions, can bevaried depending on such factors as, e.g., the concentration of the WSXligand, and the type of cells and medium.

It is contemplated that using WSX ligand to enhance cell proliferationand/or differentiation in vitro will be useful in a variety of ways. Forinstance, hematopoietic cells cultured in vitro in the presence of WSXligand can be infused into a mammal suffering from reduced levels of thecells. Also, the cultured hematopoietic cells may be used for genetransfer for gene therapy applications. Stable in vitro cultures can bealso used for isolating cell-specific factors and for expression ofendogenous or recombinantly introduced proteins in the cell. WSX ligandmay also be used to enhance cell survival, proliferation and/ordifferentiation of cells which support the growth and/or differentiationof other cells in cell culture.

The WSX receptor antibodies of the invention are also useful as affinitypurification agents. In this process, the antibodies against WSXreceptor are immobilized on a suitable support, such a Sephadex resin orfilter paper, using methods well known in the art. The immobilizedantibody then is contacted with a sample containing the WSX receptor tobe purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the WSX receptor, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the WSX receptor from theantibody.

WSX receptor antibodies may also be useful in diagnostic assays for WSXreceptor, e.g., detecting its expression in specific cells, tissues, orserum. For diagnostic applications, antibodies typically will be labeledwith a detectable moiety. The detectable moiety can be any one which iscapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin;radioactive isotopic labels, such as, e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H; or anenzyme, such as alkaline phosphatase, beta-galactosidase, or horseradishperoxidase.

Any method known in the art for separately conjugating the polypeptidevariant to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature 144:945 (1962); David et al.,Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth 40:219(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of WSX receptor in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

III Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

The disclosures of all publications, patents and patent applicationscited herein, whether supra or infra, are hereby incorporated byreference in their entirety.

EXAMPLE 1 Cloning of Human WSX Receptor

An oligonucleotide probe designated WSX.6 #1 was synthesized based uponthe T73849 EST sequence. The WSX.6 #1 probe was a 51mer having thefollowing sequence: 5′ GTCAGTCTCCCAGTTCCAGACTTGTGTGCAGTC (SEQ ID NO:45)TATGCTGTTCAGGTGCGC-3′.

The radiolabeled WSX.6 #1 probe was used to probe 1.2×10⁶ clones from arandom and oligo dT primed λgt10 fetal liver library (Clontech, PaloAlto, Calif.). Following hybridization at 42° C. overnight, the filterswere washed at 50° C. in 0.5×SSC and 0.1% NaDodSO₄ (SDS). From theinitial screen, 10 clones were selected and upon subsequent screening 5individual plaque pure clones were isolated. Of these 5 individualclones, four clones designated 1, 5, 6 and 9 were subcloned into pBSSK⁻(Stratagene) following EcoRI digestion. Sequence analysis revealed clone5 and clone 9 contained the putative initiation methionine and signalpeptide. Clone 6 (designated 6.4) contained the most 3′ end sequence andsubsequently was used for further screening.

To obtain the full length gene, clone 6.4 (fragment Nsi-Hind III) wasradiolabeled and used to screen 1.2×10⁶ clones from a λgt 10 libraryconstructed from a hepatoma Hep3B cell line. This screen resulted in 24positive clones. Following PCR analysis of the clones using λgt10primers (F and R), the four longest clones 12.1, 13.2, 22.3, and 24.3were isolated. These clones were subcloned into pBSSK⁻ using the EcoRIsite, and following examination by restriction enzyme digest, clones12.1 and 13.2 were submitted for sequencing. DNA sequencing wasperformed with the Taq dye deoxynucleotide terminator cycle sequencingkit on an automated Applied Biosystems DNA sequencer.

The assembled contiguous sequence from all the isolated clones encoded aconsensus amino terminus for the newly identified polypeptide designatedthe WSX receptor. However, sequence analysis revealed that at leastthree naturally occurring variants of the WSX receptor exist which havedifferent cytoplasmic regions. These variants appear to bedifferentially spliced at the lysine residue at position 891. Clone 6.4stops 5 amino acids after Lys 891. Clone 12.1 is different from 13.2 and6.4 following Lys 891 and encodes a putative box 2 region which isdistinct from that encoded by clone 13.2. Clone 13.2 contains apotential box 1 region and following Lys 891 encodes putative box 2 andbox 3 motifs. See, Baumann et al., Mol. Cell. Biol. 14(1):138-146(1994).

The full length WSX gene based on the clone 13.2 cytoplasmic regionputatively encodes an 1165 amino acid transmembrane protein. The 841amino acid extracellular domain (ECD) contains two WSXWS domains. TheECD is followed by a 24 amino acid transmembrane domain and a 300 aminoacid cytoplasmic region.

EXAMPLE 2 WSX Receptor Immunoadhesin

Using polymerase chain amplification, a WSX receptor immunoadhesin wascreated by engineering an in-frame fusion of the WSX receptor geneextracellular domain (WSX.ECD) with human CH2CH3(Fc)IgG (Bennett et al.,J. Biol. Chem. 266(34):23060-23067 (1991)) at the C terminus of the ECDand cloned into pBSSK⁻ (Stratagene). For expression, the WSX-Fc wasexcised with ClaI and BstEI and ligated into the pRK5.HuIF.grbhlgGGenenase I vector (Beck et al., Molecular Immunology 31(17):1335-1344(1994)), to create the plasmid pRK5.WSX-IgG Genenase I. This plasmid wastransiently transfected into 293 cells using standard calcium phosphatetransfection techniques. The transfected cells were cultured at 37° C.in 5% CO₂ in DMEM F12 50:50 supplemented with 10% FBS, 100 mM HEPES (pH7.2) and 1 mM glutamine. The WSX receptor immunoadhesin was purifiedusing a ProSepA™ protein A column.

EXAMPLE 3 Antibody Production

In order to raise antibodies against the WSX receptor, the WSX receptorimmunoadhesin of Example 2 was used to inoculate rabbits to raisepolyclonal antibodies and mice to raise monoclonal antibodies usingconventional technology.

EXAMPLE 4 Generation of a Cell Line Expressing WSX Receptor

The nucleic acid encoding full length WSX receptor variant 13.2 wasinserted in the pRKtkNeo plasmid (Holmes et al., Science 253:1278-1280(1991)). 100 μgs of the pRKtkNeo.WSX plasmid thus generated waslinearized, ethanol precipitated and resuspended in 100 μL of RPMI 1640.7×10⁶ Baf3 cells (5×10⁵/ml) were suspended in 900 μL of RPMI and addedto the linearized plasmid. Following electroporation at 325V, 1180 μFusing a BRL electroporation apparatus, the cells were plated into 15 mlsof RPMI 1640 containing 5% WEHI3B conditioned media and 15% serum. 48hours later cells were selected in 2 mg/ml G418. To obtain the Baf3/WSXcell line expressing WSX receptor variant 13.2, the G418 selected cloneswere analyzed by FACS using the rabbit polyclonal antisera raisedagainst the WSX-Fc chimeric protein as described above. The highestexpressing clone (designated E6) was sorted by FACS to maintain apopulation with a high level of WSX receptor expression.

EXAMPLE 5 Role of WSX Receptor in Cellular Proliferation

The proliferative potentials of WSX receptor variants 13.2 and 12.1 weretested by constructing human growth hormone receptor-WSX receptor(GH-WSX) fusions encoding chimeric proteins consisting of the GHreceptor extracellular and transmembrane domains and the WSX receptorvariant 13.2 or 12.1 intracellular domains. These chimeric gene fusionswere transfected into the IL-3 dependent cell line Baf3. The ability ofthe GH-WSX transfected Baf3 cells to respond to exogenous growth hormone(GH) was tested in a thymidine incorporation assay. As can be seen inFIGS. 6 and 8, the GH-WSX receptor variant 13.2 chimera was capable ofincreasing thymidine uptake in the transfected Baf3 cells, thusindicating the proliferative potential of the WSX receptor variant 13.2.However, WSX receptor variant 12.1 was unable to transmit aproliferative signal in this experiment (FIG. 8).

Materials and Methods

Recombinant PCR was used to generate the chimeric receptors containingthe extracellular and transmembrane domains of the hGH receptor and thecytoplasmic domain of either WSX receptor variant 12.1 or variant 13.2.In short, the cytoplasmic domain of either variant 12.1 or 13.2beginning with Arg at amino acid 866 and extending down to amino acid958 or amino acid 1165 respectively, was fused in frame, by sequentialPCR, to the hGH receptor extracellular and transmembrane domainbeginning with Met at amino acid 18 and extending down to Arg at aminoacid 274. The GH-WSX chimera was constructed by first using PCR togenerate the extracellular and transmembrane domain of the human GHreceptor. The 3′ end primer used for this PCR contained 20 nucleotidesat the 5′ end of the primer corresponding to the first 20 nucleotides ofthe WSX cytoplasmic domain. The 3′ end of the chimera was generatedusing PCR where the 5′ end primer contained the last 19 nucleotides ofthe human GH receptor transmembrane domain. To generate the full lengthchimera, the 5′ end of the human GH receptor product was combined withthe 3′ end WSX receptor cytoplasmic PCR product and subsequentlyamplified to create a fusion of the two products.

This chimeric fusion was digested with ClaI and XbaI and ligated topRKtkNeo (Holmes et al., Science 253:1278-1280 (1991)) to create thechimeric expression vector. The IL-3 dependent cell line Baf3 was thenelectroporated with this hGH/WSX chimeric expression vector.

Briefly, 100 μg of the pRKtkNeo/GH.WSX plasmid was linearized, ethanolprecipitated and resuspended in 100 μL of RPMI 1640. 7×10⁶ Baf3 cells(5×10⁵/ml) were suspended in 900 μL of RPMI and added to the linearizedplasmid. Following electroporation at 325V, 1180 μF using a BRLelectroporation apparatus, the cells were plated into 15 mls of RPMI1640 containing 5% wehi conditioned media and 15% serum. 48 hours later,cells were selected in 2 mg/ml G418.

To obtain the Baf3/GH.WSX cell lines, the G418 selected cells were FACSsorted using an anti-human GH Mab (3B7) at 1 μg/ml. The top 10%expressing cells were selected and expanded.

EXAMPLE 6 Expression Analysis of the WSX Receptor

The expression profile of the WSX receptor was initially examined byNorthern analysis. Northern blots of human fetal or adult tissue mRNAwere obtained from Clontech (Palo Alto, Calif.). A transcript ofapproximately 6 kb was detected in human fetal lung, liver and kidney.In the adult, low level expression was detected in a variety of tissuesincluding liver, placenta, lung skeletal muscle, kidney, ovary, prostateand small intestine.

PCR analysis of human cord blood identified transcripts in CD34⁺subfraction. By PCR analysis, all three variants of the WSX receptorwere present in CD34⁺ cells. The CD34⁻ subfraction appeared negative bythis same PCR analysis.

By PCR analysis, both the 6.4 variant and 13.2 variant were evident inthe AA4⁺Sca⁺Kit⁺ (flASK) cell population isolated from the mid-gestationfetal liver as described in Zeigler et al., Blood 84:2422-2430 (1994).No clones containing the 12.1 variant cytoplasmic tail have beenisolated from murine tissues.

Human B cells isolated from peripheral blood using anti-CD19/20antibodies were also positive for short form (6.4 variant) and long form(13.2 variant) receptor mRNA expression.

The WSX receptor appears to be expressed on both progenitor and moremature hematopoietic cells.

EXAMPLE 7 Cloning of Murine WSX Receptor

The human WSX receptor was used as a probe to isolate murine WSXreceptor. The pRKtkNeo.WSX plasmid of Example 4 was digested using Ssp1.This Ssp1 fragment (1624 bps) was isolated, and radiolabelled, and usedto screen a murine liver λgt10 library (Clontech). This resulted in 4positive clones which were isolated and sequenced after sub-cloning intopBSSK⁻ via EcoRI digestion. The resultant clones, designated 1, 2, 3, 4showed homology to the extracellular domain of the human WSX receptor;the contiguous sequences resulting from these clones extended from theinitiation methionine to tryptophan at position 783. The overallsimilarity of human WSX receptor and murine WSX receptor is 73% overthis region of the respective extracellular domains (see FIGS. 4A-D).

EXAMPLE 8 The Role of WSX Receptor in Hematopoietic Cell Proliferation

The presence of the WSX receptor in the enriched human stem cellpopulation CD34⁺ from cord blood is indicative of a potential role forthis receptor in stem cell/progenitor cell proliferation. Theproliferation of CD34⁺ human blood cells in methylcellulose media (StemCell Technologies) was determined in the presence or absence of WSXreceptor antisense oligonucleotides. These experiments were alsorepeated in the murine hematopoietic system using AA4⁺Sca⁺Kit⁺ stemcells from the murine fetal liver. In both instances, the antisenseoligonucleotides statistically significantly inhibited colony formationfrom the hematopoietic progenitor cells. See Table 1 below. Theanti-proliferative effects were most pronounced using the −20 antisenseand the +85 antisense oligonucleotide constructs. This inhibition wasnot lineage specific to any particular myeloid lineage that resultedfrom the progenitor expansion. The principal effect of the antisenseoligonucleotides was a reduction of overall colony numbers. The size ofthe individual colonies was also reduced.

Antisense oligonucleotide experiments using both human and murine stemcells demonstrated an inhibition of myeloid colony formation. Although,the reduction in myelopoiesis observed in these assays could beprevented by the additional inclusion of G-CSF and GM-CSF in the culturemedium. These data serve to illustrate the redundancy of cytokine actionin the myelopoietic compartment. TABLE 1 AVG. % INHI- EXPERIMENT OLIGOCOLONY # BITION Human Cord Blood (KL) (−20)AS 32 (−20)S 100 70 (−20)SCR114 (+85)AS 80 (+85)S 123 38 (+85)SCR 138 Control 158 Human Cord Blood(−20)AS 78 (IL-3, IL-6, KL) (−20)S 188 54 (−20)SCR 151 (+85)AS 167(+85)S 195 18 (+85)SCR 213 Control 266 Human Cord Blood (KL) (−20)AS 42(−20)S 146 69 (−20)SCR 121 (+85)AS 123 (+85)S 162 23 (+85)SCR 156Control 145 Murine Fetal Liver (KL) (+84)AS 33 (+84)S 86 54 (+84)SCR 57(−20)AS 27 (−20)S 126 71 (−20)SCR 60 (−99)AS 109 (−99)S 93 0 (−99)SCR109 Control 121 Murine Fetal Liver (KL) (−213)AS 51 (−213)S 60 10(−213)SCR 53 (+211)AS 58 (+211)S 54 3 (+211)SCR 66 Control 59Materials and Methods

Human stem cells: Human umbilical cord blood was collected inPBS/Heparin (1000 μ/ml). The mononuclear fraction was separated using adextran gradient and any remaining red blood cells lysed in 20 mM NH₄Cl. CD34⁺ cells were isolated using CD34⁺ immunomagnetic beads(Miltenyi, Calif.). These isolated CD34⁺ cells were found to be 90-97%CD34⁺ by FACS analysis.

Murine stem cells: Midgestation fetal liver were harvested andpositively selected for the AA4⁻ antigen by immune panning. The AA4⁻positive fraction was then further enriched for stem cell content byFACS isolation of the AA4⁺Sca⁺Kit⁺ fraction.

Antisense experiments: Oligodeoxynucleotides were synthesized againstregions of the human or murine WSX receptors. For each oligonucleotidechosen, antisense (AS), sense (S) and scrambled (SCR) versions weresynthesized (see FIG. 7). + or − indicates position relative theinitiation methionine of the WSX receptor. CD34⁺ or AA4⁺Sca⁺Kit⁺ cellswere incubated at a concentration of 10³/ml in 50:50 DMEM/F12 mediasupplemented with 10% FBS, L-glutamine, and GIBCO™ lipid concentratecontaining either sense, antisense or scrambled oligonucleotides at aconcentration of 70 μg/ml. After 16 hours, a second aliquot of therespective oligonucleotide was added (35 μg/ml) and the cells incubatedfor a further 6 hours.

Colony assays: 5000 cells from each of the above conditions werealiquoted into 5 ml of methylcellulose (Stem Cell Technologies)containing kit ligand (KL) (25 ng/ml), interleukin-3 (IL-3) (25 ng/ml)and interleukin-6 (IL-6) (50 ng/ml). The methylcellulose cultures werethen incubated at 37° C. for 14 days and the resultant colonies countedand phenotyped. All assays were performed in triplicate.

EXAMPLE 9 WSX Receptor Variant 13.2 is a Receptor for OB Protein

The WSX receptor variant 13.2 has essentially the same amino acidsequence as the recently cloned leptin (OB) receptor. See Tartaglia etal., Cell 83:1263-1271 (1995). OB protein was able to stimulatethymidine incorporation in Baf3 cells transfected with WSX receptorvariant 13.2 as described in Example 4 (See FIG. 9).

OB protein expression in hematopoietic cells was studied.Oligonucleotide primers designed specifically against the OB proteinillustrated the presence of this ligand in fetal liver and fetal brainas well as in two fetal liver stromal cell lines, designated 10-6 and7-4. Both of these immortalized stromal cell lines have beendemonstrated to support both myeloid and lymphoid proliferation of stemcell populations (Zeigler et al., Blood 84:2422-2430 (1994)).

EXAMPLE 10 Role of OB Protein in Hematopoiesis

To examine the hematopoietic activity of OB protein, a variety of invitro assays were performed.

Murine fetal liver flASK stem cells were isolated from themidgestational fetal liver as described in Zeigler et al., Blood84:2422-2430 (1994) and studied in stem cell suspension culture ormethylcellulose assays.

For the stem cell suspension cultures, twenty thousand of the fLASKcells were seeded in individual wells in a 12 well format in DMEM4.5/F12 media supplemented with 10% heat inactivated fetal calf serum(Hyclone, Logan, Utah) and L-glutamine. Growth factors were added at thefollowing concentrations: kit ligand (KL) at 25 ng/mL, interleukin-3(IL-3) at 25 ng/mL, interleukin-6 (IL-6) at 50 ng/mL, G-CSF at 100ng/mL, GM-CSF at 100 ng/mL, EPO at 2U/mL, interleukin-7 (IL-7) at 100ng/mL (all growth factors from R and D Systems, Minneapolis, Minn.). OBprotein was added at 100 ng/mL unless indicated otherwise. RecombinantOB protein was produced as described in Levin et al., Proc. Natl. Acad.Sci. (USA) 93:1726-1730 (1996).

In keeping with its ability to transduce a proliferative signal in Baf3cells (see previous Example), OB protein dramatically stimulated theexpansion of flASK cells grown in suspension culture in the presence ofkit ligand (FIG. 10A). The addition of OB protein alone to thesesuspension cultures was unable to effect survival of the hematopoieticstem cells (HSCs). When a variety of hematopoietic growth factors insuspension culture assays were tested, the main synergy of OB proteinappeared to be with KL, GM-CSF and IL-3 (Table 2). No preferentialexpansion of any particular lineage was observed from cytospin analysisof the resultant cultures. TABLE 2 Factor KL KL + OB protein OB proteinN/A 128 +/− 9  192 +/− 13 G-CSF 131 +/− 3 177 +/− 8 30 +/− 5 GM-CSF 148+/− 4 165 +/− 6 134 +/− 10 IL-3 189 +/− 7 187 +/− 4  144+/−    IL-6 112+/− 4 198 +/− 5 32 +/− 3 EPO 121 +/− 3 177 +/− 8 30 +/− 6 IL-3 & IL-6 112 +/− 12 198 +/− 7 32 +/− 7flASK stem cells were isolated. Twenty thousand cells were plated insuspension culture with the relevant growth factor combination. Cellswere harvested and counted after 7 days. Cell numbers are presented×10³. Assays were performed in triplicate and repeated in twoindependent experiments.

Methylcellulose assays were performed as previously described (Zeiger etal., supra). Briefly, methylcellulose colony assays were performed using“complete” methylcellulose or pre-B methylcellulose medium (Stem CellTechnologies, Vancouver, British Columbia, Canada) with the addition of25 ng/mL KL (R and D Systems, Minneapolis, Minn.). Cytospin analyses ofthe resultant colonies were performed as previously described in Zeigleret al.

When these methylcellulose assays were employed, OB protein augmentedmyeloid colony formation and dramatically increased lymphoid anderythroid colony formation (FIGS. 10B and 10C) which demonstrates thatOB protein can act on very early cells of the hematopoietic lineage.Importantly, the hematopoietic activity of OB protein was not confinedto fetal liver stem cells, the murine bone marrow stem cell population;Lin^(lo)Sca⁺ also proliferated in response to OB protein (KL: 5 foldexpansion, KL and OB protein: 10 fold expansion).

Further hematopoietic analysis of the role of the WSX receptor wascarried out by examining hematopoietic defects in the db/db mouse.

These defects were assessed by measuring the proliferative potential ofdb/db homozygous mutant marrow. Under conditions favoring either myeloid(Humphries et al., Proc. Natl. Acad. Sci. (USA) 78:3629-3633 (1981)) orlymphoid (McNiece et al., J. Immunol. 146:3785-90 (1991)) expansion, thecolony forming potential of the db/db marrow was significantly reducedwhen compared to the wild-type control marrow (FIG. 11). This wasparticularly evident when the comparison was made under pre-Bmethylcellulose conditions where KL and IL-7 are used to drivelymphopoiesis (McNiece et al., supra). Corresponding analysis of thecomplementary mouse mutation ob/ob, which is deficient in the productionof OB protein (Zhang et al., Nature 372:425-431 (1994)), also indicatedthat the lymphoproliferative capacity is compromised in the absence of afunctional OB protein signalling pathway (FIG. 11). However, thisreduction was less than the reduction observed using db/db marrow.

Analysis of the cellular profile of the db/db and wild-type marrowrevealed significant differences between the two. Overall cellularity ofthe db/db marrow was unchanged. However, when various B cell populationsin the db/db marrow were examined, both decreased levels of B220⁺ andB220⁺/CD43⁺ cells were found. B220+cells represent all B cell lineageswhile CD43 is considered to be expressed preferentially on the earliestcells of the B cell hierarchy (Hardy et al., J. Exp. Med. 173:1213-25(1991)). No differences were observed between the CD4/CD8 stainingprofiles of the two groups. The TER119 (a red cell lineage marker)population was increased in the db/db marrow (FIG. 12A).

Comparison of the spleens from the two groups revealed a significantdecrease in both tissue weight and cellularity of the db/db micecompared to the homozygote misty gray controls (0.063±0.009 g vs.0.037±0.006 g and 1.10×10⁷±1×10⁴ vs. 4.3×10⁶±10³ cells >p0.05). Thisdecreased cellularity in the db spleen was reflected in a markedreduction in TER119 staining (FIG. 12B). This result appears to confirmthe synergy demonstrated between OB protein and EPO and points to a rolefor OB protein in the regulation of erythropoiesis.

Examination of the hematopoietic compartment of the db/db mouse in vivodemonstrated a significant reduction in peripheral blood lymphocyteswhen compared to heterozygote or wild-type controls. Db/db mice fail toregulate blood glucose levels and become diabetic at approximately 6-8weeks of age; therefore, peripheral blood counts as the animals maturedwere followed.

For procurement of blood samples, prior to the experiment and at timepoints throughout the study, 40 μL of blood was taken from the orbitalsinus and immediately diluted into 10 mL of diluent to prevent clotting.The complete blood count from each blood sample was measured on aSerrono Baker system 9018 blood analyzer within 60 min. of collection.Only half the animals in each dose group were bled on any given day,thus, each animal was bled on alternate time points. Blood glucoselevels were measured in orbital sinus blood samples using One Touchglucose meters and test strips (Johnson and Johnson). The results ofthis experiment are shown in FIGS. 13A-C.

This analysis demonstrated that peripheral blood lymphocytes aresignificantly reduced at all time points compared to control animals andthat the peripheral lymphocyte population of the db/db mouse does notchange significantly with age. FACS analysis revealed that the decreasedlymphocyte population represented a decrease in both B220⁺ cells andCD4/CD8 cells. Both erythrocyte and platelets are at wild-type levelsthroughout all time periods examined. The peripheral blood lymphocytelevels in ob/ob homozygous mutant mice were unchanged from wild-typecontrols.

Hematopoietic analysis of the db/db mouse can be complicated by theonset of diabetes. Therefore, the impact of high glucose levels onlymphopoiesis was examined by comparing the peripheral blood profilesand blood glucose levels in two other diabetic models, the glucokinaseknockout heterozygote mouse (Grupe et al., Cell 83:69-78 (1995)) and theIFN-α transgenic mouse (Stewart et al., Science 260:1942-6 (1993)).Comparison of peripheral lymphocytes and blood glucose in db/db mice,their appropriate controls and the high glucose models illustrated norelationship between blood-glucose and lymphocyte counts (FIG. 14).These results suggest therefore that the lymphoid defects observed inthe db/db mouse are directly attributed to the hematopoietic function ofthe OB protein signalling pathway.

To test the capacity of the db/db hematopoietic compartment to respondto challenge, the db/db mice and controls were subjected to sub-lethalirradiation C57BLKS/J db/db, C57BLKS/Jm⁺/db, and C57BLKS/J⁺m/⁺m micewere subjected to sub-lethal whole body irradiation (750 cGy, 190cGy/min) as a single dose from a ¹³⁷CS source. Ten animals were used perexperimental group. The kinetics of hematopoietic recovery were thenfollowed by monitoring the peripheral blood during the recovery phase.This experiment illustrated the inability of the db/db hematopoieticsystem to fully recover the lymphopoietic compartment of the peripheralblood 35 days post-irradiation. Platelet levels in these mice followedthe same recovery kinetics as controls, however the reduction inerythrocytes lagged behind controls by 7-10 days. This finding mayreflect the increased TER 119 population found in the marrow of thedb/db mice (FIG. 12A).

Materials and Methods

Bone marrow, spleens and peripheral blood was harvested from thediabetic mouse strains: C57BLKS/J db/db (mutant), C57BLKS/J m+/db (leanheterozygote control littermate), C57BLKS/J+m/+m (lean homozygote mistygray coat control littermate) and the obese mouse strains:C57BL/6J−ob/ob (mutant) and the C57BL/6J−ob/+ (lean littermate control).All strains from the Jackson Laboratory, Bar Harbor, Me. A minimum offive animals were used per experimental group. Femurs were flushed withHank's balanced salt solution (HBSS) plus 2% FCS and a single cellsuspension was made of the bone marrow cells. Spleens were harvested andthe splenic capsule was ruptured and filtered through a nylon mesh.Peripheral blood was collected through the retro-orbital sinus inphosphate buffered saline (PBS) with 10 U/mL heparin and 1 mmol EDTA andprocessed as previously described. The bone marrow, splenocytes andperipheral blood were then stained with the monoclonal antibodiesagainst the following antigens: B220/CD45R (Pan B cell) FITC antimouse,TER-119/erythroid cell R-PE antimouse, CD4 (L3T4), FITC antimouse, CD8(Ly 3.2), FITC antimouse, and sIgM (Igh-6b), FITC antimouse (Allmonoclonals from Pharmigen, San Diego, Calif.). The appropriate isotypecontrols were included in each experiment. For methylcellulose assays,the bone marrow from five animals per group was pooled and 100,000 cellaliquots from each group used for each assay point.

EXAMPLE 11 Expression of OB-Immunoadhesin

Using protein engineering techniques, the human OB protein was expressedas a fusion with the hinge, CH2 and CH3 domains of IgG1. DNA constructsencoding the chimera of the human OB protein and IgG1 Fc domains weremade with the Fc region clones of human IgG1. Human OB cDNA was obtainedby PCR from human fat cell dscDNA (Clontech Buick-Clone cDNA product).The source of the IgG1 cDNA was the plasmid pBSSK-CH2CH3. The chimeracontained the coding sequence of the full length OB protein (amino acids1-167 in FIG. 16) and human IgG1 sequences beginning at aspartic acid216 (taking amino acid 114 as the first residue of the heavy chainconstant region (Kabat et al., Sequences of Proteins of ImmunologicalInterest 4th ed. (1987)), which is the first residue of the IgG1 hingeafter the cysteine residue involved in heavy-light chain bonding, andending with residues 441 to include the CH2 and CH3 Fc domains of IgG1.There was an insert of codons for three amino acids (GlyValThr) betweenthe OB protein and IgG1 coding sequences. If necessary, this shortlinker sequence can easily be deleted, for example by site directeddeletion mutagenesis, to create an exact junction between the codingsequences of the OB protein and the IgG1 hinge region. The codingsequence of the OB-IgG1 immunoadhesin was subcloned into the pRK5-basedvector pRK5tk-neo which contains a neomycine selectable marker, fortransient expression in 293 cells using the calcium phosphate technique(Suva et al., Science 237:893-896 (1987)). 293 cells were cultured inHAM's: Low Glucose DMEM medium (50:50), containing 10% FBS and 2 mML-Gln. For purification of OB-IgG1 chimeras, cells were changed to serumfree production medium PS24 the day after transfection and mediacollected after three days. The culture media was filtered.

The filtered 293 cell supernatant (400 ml) containing recombinant humanOB-IgG1 was made 1 mM in phenylmethylsulfonyl fluoride and 2 μg/ml inaprotinin. This material was loaded at 4° C. onto a 1×4.5 cm Protein Aagarose column (Pierce catalog # 20365) equilibrated in 100 mM HEPES pH8. The flow rate was 75 ml/h. Once the sample was loaded, the column waswashed with equilibration buffer until the A₂₈₀ reached baseline. TheOB-IgG1 protein was eluted with 3.5 M MgCl₂+2% glycerol (unbuffered) ata flow rate of 15 ml/h. The eluate was collected with occasional mixinginto 10 ml of 100 mM HEPES pH 8 to reduce the MgCl₂ concentration byapproximately one-half and to raise the pH. The eluted protein was thendialyzed into phosphate buffered saline, concentrated, sterile filteredand stored either at 4° C. or frozen at −70° C. The OB-IgG1immunoadhesin prepared by this method is estimated by SDS-PAGE to begreater than 90% pure.

EXAMPLE 12 Preparation of PEG-OB

The PEG derivatives of the human OB protein were prepared by reaction ofhOB protein purified by reverse phase chromatography with a succinimidylderivative of PEG propionic acid (SPA-PEG) having a nominal molecularweight of 10 kD, which had been obtained from Shearwater Polymers, Inc.(Huntsville, Ala.). After purification of the hOB protein by reversephase chromatography, an approximately 1-2 mg/ml solution of the proteinin 0.1% trifluoroacetic acid and approximately 40% acetonitrile, wasdiluted with ⅓ to ½ volume of 0.2 M borate buffer and the pH adjusted to8.5 with NaOH. SPA-PEG was added to the reaction mixture to make 1:1 and1:2 molar ratios of protein to SPA-PEG and the mixture was allowed toincubate at room temperature for one hour. After reaction andpurification by gel electrophoresis or ion exchange chromatography, thesamples were extensively dialyzed against phosphate-buffered saline andsterilized by filtration through a 0.22 micron filter. Samples werestored at 4° C. Under these conditions, the PEG-hOB resulting from the1:1 molar ratio protein to SPA-PEG reaction consisted primarily ofmolecules with one 10 kD PEG attached with minor amounts of the 2PEG-containing species. The PEG-hOB from the 1:2 molar reactionconsisted of approximately equal amounts of 2 and 3 PEGs attached tohOB, as determined by SDS gel electrophoresis. In both reactions, smallamounts of unreacted protein were also detected. This unreacted proteincan be efficiently removed by the gel filtration or ion exchange stepsas needed. The PEG derivatives of the human OB protein can also beprepared essentially following the aldehyde chemistry described in EP372,752 published Jun. 13, 1990.

1. A method for enhancing proliferation or differentiation of a cell ofthe hematopoietic lineage comprising an OB receptor having a WSX motif,said method comprising administering to the cell an amount of OB proteinwhich is effective for enhancing proliferation or differentiation of thecell, with the proviso that a further cytokine is not concurrentlyadministered to the cell, wherein a sequence of said OB protein sharesat least 84% sequence homology with a mouse OB protein sequence, sharesat least 84% sequence homology with a human OB protein sequence, orshares at least 84% sequence homology with both a human and a mouse OBprotein sequences.
 2. The method of claim 1, wherein the sequence ofsaid OB protein is at least 90% identical to the mouse OB proteinsequence.
 3. The method of claim 1, wherein the sequence of said OBprotein is at least 95% identical to the mouse OB protein sequence. 4.The method of claim 1, wherein the sequence of said OB protein is atleast 99% identical to the mouse OB protein sequence.
 5. The method ofclaim 1, wherein the sequence of said OB protein shares at least 84%sequence homology with the human OB protein sequence.
 6. The method ofclaim 1, wherein the sequence of said OB protein is at least 90%identical to the human OB protein sequence.
 7. The method of claim 1,wherein the sequence of said OB protein is at least 95% identical to thehuman OB protein sequence.
 8. The method of claim 1, wherein a sequenceof said OB protein is at least 99% identical to the human OB proteinsequence.
 9. The method of claim 1, wherein said OB protein is encodedby a nucleic acid sequence that hybridizes to a mouse nucleic acidsequence, wherein said mouse nucleic acid sequence encodes a mouse OBprotein.
 10. A method for enhancing proliferation or differentiation ofa cell of the hematopoietic lineage comprising an OB receptor having aWSX motif, said method comprising administering to the cell an amount ofOB protein which is effective for enhancing proliferation ordifferentiation of the cell, with the proviso that a further cytokine isnot concurrently administered to the cell, wherein said OB protein isencoded by a nucleic acid sequence that hybridizes to a human nucleicacid sequence, wherein said human nucleic acid sequence encodes a humanOB protein, and wherein said hybridization occurs under moderatelystringent conditions.
 11. A method for repopulating blood cells in amammal comprising administering to the mammal a therapeuticallyeffective amount of OB protein, with the proviso that a further cytokineis not concurrently administered to the mammal to repopulate blood cellswhen the OB protein is administered to the mammal, wherein a sequence ofsaid OB protein shares at least 84% sequence homology with a mouse OBprotein sequence, shares at least 84% sequence homology with a human OBprotein sequence, or shares at least 84% sequence homology with both ahuman and a mouse OB protein sequences.
 12. The method of claim 11,wherein the sequence of said OB protein is at least 90% identical to themouse OB protein sequence.
 13. The method of claim 11, wherein thesequence of said OB protein is at least 95% identical to the mouse OBprotein sequence.
 14. The method of claim 11, wherein the sequence ofsaid OB protein is at least 99% identical to the mouse OB proteinsequence.
 15. The method of claim 11, wherein the sequence of said OBprotein shares at least 84% sequence homology with the human OB proteinsequence.
 16. The method of claim 11, wherein the sequence of said OBprotein is at least 90% identical to the human OB protein sequence. 17.The method of claim 11, wherein the sequence of said OB protein is atleast 95% identical to the human OB protein sequence.
 18. The method ofclaim 11, wherein a sequence of said OB protein is at least 99%identical to the human OB protein sequence.
 19. The method of claim 11,wherein said OB protein is encoded by a nucleic acid sequence thathybridizes to a mouse nucleic acid sequence, wherein said mouse nucleicacid sequence encodes a mouse OB protein.
 20. The method of claim 11,wherein said OB protein is encoded by a nucleic acid sequence thathybridizes to a human nucleic acid sequence, wherein said human nucleicacid sequence encodes a human OB protein, and wherein said hybridizationoccurs under moderately stringent conditions.